Hepatitis c virus replicons and replicon enhanced cells

ABSTRACT

The present invention features nucleic acid containing one or more adaptive mutations, and HCV replicon enhanced cells. Adaptive mutations are mutations that enhance HCV replicon activity. HCV replicon enhanced cells are cells having an increased ability to maintain an HCV replicon.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Ser. No.60/263,479, filed Jan. 23, 2001, hereby incorporated by referenceherein.

BACKGROUND OF THE INVENTION

[0002] The references cited in the present application are not admittedto be prior art to the claimed invention.

[0003] It is estimated that about 3% of the world's population areinfected with the Hepatitis C virus (HCV). (Wasley, et al. 2000. Semin.Liver Dis. 20, 1-16.) Exposure to HCV results in an overt acute diseasein a small percentage of cases, while in most instances the virusestablishes a chronic infection causing liver inflammation and slowlyprogresses into liver failure and cirrhosis. (Iwarson, 1994. FEMSMicrobiol. Rev. 14, 201-204.) In addition, epidemiological surveysindicate an important role of HCV in the pathogenesis of hepatocellularcarcinoma. (Kew, 1994. FEMS Microbiol. Rev. 14, 211-220, Alter, 1995.Blood 85, 1681-1695.)

[0004] The HCV genome consists of a single strand RNA of about 9.5 kb inlength, encoding a precursor polyprotein of about 3000 amino acids.(Choo, et al., 1989. Science 244, 362-364, Choo, et al., 1989. Science244, 359-362, Takamizawa, et al., 1991. J. Virol. 65, 1105-1113.) TheHCV polyprotein contains the viral proteins in the order:C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.

[0005] Individual viral proteins are produced by proteolysis of the HCVpolyprotein. Host cell proteases release the putative structuralproteins C, E1, E2, and p7, and create the N-terminus of NS2 at aminoacid 810. (Mizushima, et al., 1994. J. Virol. 68, 2731-2734, Hijikata,et al., 1993. P.N.A.S. USA 90, 10773-10777.)

[0006] The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5Bpresumably form the virus replication machinery and are released fromthe polyprotein. A zinc-dependent protease associated with NS2 and theN-terminus of NS3 is responsible for cleavage between NS2 and NS3.(Grakoui, et al, 1993. J. Virol. 67, 1385-1395, Hijikata, et al., 1993.P.N.A.S. USA 90, 10773-10777.) A distinct serine protease located in theN-terminal domain of NS3 is responsible for proteolytic cleavages at theNS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions.(Barthenschlager, et al., 1993. J. Virol. 67, 3835-3844, Grakoui, etal., 1993 . Proc. Natl. Acad. Sci. USA 90, 10583-10587, Tomei, et al.,1993. J. Virol. 67, 4017-4026.) NS4A provides a cofactor for NS3activity. (Failla, et al., J. Virol. 1994. 68, 3753-3760, De Francesco,et al., U.S. Pat. No. 5,739,002.) NS5A is a highly phosphorylatedprotein concurring interferon resistance. (De Francesco, et al., 2000.Semin Liver Dis., 20(1), 69-83, Pawlotsky, 1999. J. Viral Hepat. Suppl.1, 47-48.) NS5B provides an RNA polymerase. (De Francesco, et al.,International Publication Number WO 96/37619, Behrens, et al., 1996.EMBO 15, 12-22, Lohmann, et al., 1998. Virology 249, 108-118.)

[0007] Lohmann, et al., Science 285, 110-113, 1999, illustrates theability of a biscistronic HCV replicon to replicate in a hepatoma cellline. The biscistonic HCV replicon contained a neomycin cistron and anNS2-NS5B or an NS3-NS5B cistron. “NS2-NS5B” refers to aNS2-NS3-NS4A-NS4B-NS5A-NS5B polyprotein. “NS3NS5B” refers to aNS3-NS4A-NS4B-NS5A-NS5B polyprotein.

[0008] Bartenschlager, European Patent Application 1 043 399, publishedOct. 11, 2000 (not admitted to be prior art to the claimed invention),describes a cell culture system for autonomous HCV RNA replication andprotein expression. Replication and protein expression is indicated tooccur in sufficiently large amounts for quantitative determination.European Patent Application 1 043 399 indicates that prior cell lines orprimary cell cultures infected with HCV do not provide favorablecircumstances for detecting HCV replication.

SUMMARY OF THE INVENTION

[0009] The present invention features nucleic acid containing one ormore adaptive mutations, and HCV replicon enhanced cells. Adaptivemutations are mutations that enhance HCV replicon activity. HCV repliconenhanced cells are cells having an increased ability to maintain an HCVreplicon.

[0010] An HCV replicon is an RNA molecule able to autonomously replicatein a cultured cell and produce detectable levels of one or more HCVproteins. The basic subunit of an HCV replicon encodes for a HCVNS3-NS5B polyprotein along with a suitable 5′ UTR-partial core (PC)region and 3′ UTR. The 5′ UTR-PC region is made up of a 5′ UTR regionand about 36 nucleotides of the beginning of the core. Additionalregions may be present including those coding for HCV proteins orelements such as the complete core, E1, E2, p7 or NS2; and those codingfor other types of proteins or elements such as a encephalomyocarditisvirus (EMCV) internal ribosome entry site (IRES), a reporter protein ora selection protein.

[0011] The present application identifies different adaptive mutationsthat enhance HCV replicon activity. Enhancing replicon activity bringsabout at least one of the following: an increase in replicon maintenancein a cell, an increase in replicon replication, and an increase inreplicon protein expression.

[0012] Adaptive mutations are described herein by identifying thelocation of the adaptive mutation with respect to a reference sequencepresent in a particular region. Based on the provided referencesequence, the same adaptive mutation can be produced in correspondinglocations of equivalent regions having an amino acid sequence differentthan the reference sequence. Equivalent regions have the same functionor encode for a polypeptide having the same function.

[0013] Replicon enhanced cells are a preferred host for the insertionand expression of an HCV replicon. Replicon enhanced cells are initiallyproduced by creating a cell containing a HCV replicon and then curingthe cell of the replicon. The term “replicon enhanced cell” includescells cured of HCV replicons and progeny of such cells.

[0014] Thus, a first aspect of the present invention describes a nucleicacid molecule comprising at least one of the following regions: analtered NS3 encoding region, an altered NS5A encoding region, and analtered EMCV IRES region. The altered region contains one or moreadaptive mutations. Reference to the presence of particular adaptivemutation(s) does not exclude other mutations or adaptive mutations frombeing present. Adaptive mutations are described with reference to eitheran encoded amino acid sequence or a nucleic acid sequence.

[0015] A nucleic acid molecule can be single-stranded or part of adouble strand, and can be RNA or DNA. Depending upon the structure ofthe nucleic acid molecule, the molecule may be used as a replicon or inthe production of a replicon. For example, single-stranded RNA havingthe proper regions can be a replicon, while double-stranded DNA thatincludes the complement of a sequence coding for a replicon or repliconintermediate may useful in the production of the replicon or repliconintermediate.

[0016] Preferred nucleic acid molecules are those containing region(s)from SEQ. ID. NOs. 1, 2, or 3, or the RNA version thereof, with one ormore adaptive mutations. Reference to “the RNA version thereof”indicates a ribose backbone and the presence of uracil instead ofthymine.

[0017] The presence of a region containing an adaptive mutationindicates that at least one such region is present. In differentembodiments, for example, adaptive mutations described herein arepresent at least in the NS3 region, in the NS5A region, in the NS3 andNS5A regions, in the EMCV IRES and NS3 regions, in the EMCV and NS5Aregions, and in the ECMV IRES, NS3 and NS5A regions.

[0018] Another aspect of the present invention describes an expressionvector comprising a nucleotide sequence of an HCV replicon or repliconintermediate coupled to an exogenous promoter. Reference to a nucleotidesequence “coupled to an exogenous promoter” indicates the presence andpositioning of an RNA promoter such that it can mediate transcription ofthe nucleotide sequence and that the promoter is not naturallyassociated with the nucleotide sequence being transcribed. Theexpression vector can be used to produce RNA replicons.

[0019] Another aspect of the present invention describes a recombinanthuman hepatoma cell. Reference to a recombinant cell includes aninitially produced cell and progeny thereof.

[0020] Another aspect of the present invention describes a method ofmaking a HCV replicon enhanced cell. The method involves the steps of:(a) introducing and maintaining an HCV replicon into a cell and (b)curing the cell of the HCV replicon.

[0021] Another aspect of the present invention describes an HCV repliconenhanced cell made by a process comprising the steps of: (a) introducingand maintaining an HCV replicon into a cell and (b) curing the cell ofthe HCV replicon.

[0022] Another aspect of the present invention describes a method ofmaking a HCV replicon enhanced cell comprising an HCV replicon. Themethod involves (a) introducing and maintaining a first HCV repliconinto a cell, (b) curing the cell of the replicon, and (c) introducingand maintaining a second replicon into the cured cell, where the secondreplicon may be the same or different as the first replicon.

[0023] Another aspect of the present invention describes an HCV repliconenhanced cell containing a HCV replicon made by the process involvingthe step of introducing an HCV replicon into an HCV replicon enhancedcell. The HCV replicon introduced into the HCV replicon enhanced cellmay be the same or different than the HCV replicon used to produce theHCV replicon enhanced cell. In a preferred embodiment, the HCV repliconintroduced into an HCV replicon enhanced cell is the same replicon aswas used to produce the enhanced cell.

[0024] Another aspect of the present invention describes a method ofmeasuring the ability of a compound to affect HCV activity using an HCVreplicon comprising an adaptive mutation described herein. The methodinvolves providing a compound to a cell comprising the HCV replicon andmeasuring the ability of the compound to affect one or more repliconactivities as a measure of the effect on HCV activity.

[0025] Another aspect of the present invention describes a method ofmeasuring the ability of a compound to affect HCV activity using an HCVreplicon enhanced cell that comprises an HCV replicon. The methodinvolves providing a compound to the cell and measuring the ability ofthe compound to effect one or more replicon activities as a measure ofthe effect on HCV activity.

[0026] Other features and advantages of the present invention areapparent from the additional descriptions provided herein including thedifferent examples. The provided examples illustrate differentcomponents and methodology useful in practicing the present invention.The examples do not limit the claimed invention. Based on the presentdisclosure the skilled artisan can identify and employ other componentsand methodology useful for practicing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGS. 1A-1G illustrate the nucleic acid sequence for thepHCVNeo.17 coding strand (SEQ. ID. NO. 3). The different regions ofpHCVNeo.17 are provided as follows:

[0028] 1-341: HCV 5′ non-translated region, drives translation of thecore-neo fusion protein;

[0029] 342-1181: Core-neo fusion protein, selectable marker;

[0030] 1190-1800: Internal ribosome entry site of theencephalomyocarditis virus, drives translation of the HCV NS region;

[0031] 1801-7755: HCV polyprotein from non-structural protein 3 tonon-structural protein SB;

[0032] 1801-3696: Non-structural protein 3 (NS3), HCV NS3protease/helicase;

[0033] 3697-3858: Non-structural protein 4A (NS4A), NS3 proteasecofactor;

[0034] 3859-4641: Non-structural protein 4B (NS4B);

[0035] 4642-5982: Non-structural protein 5A (NS5A);

[0036] 5983-7755: Non-structural protein 5B (NS5B); RNA-dependent RNApolymerase

[0037] 7759-7989: HCV 3′ non-translated region; and

[0038] 7990-10690 plasmid sequences comprising origin of replication,beta lactamase coding sequence, and T7 promoter.

DETAILED DESCRIPTION OF THE INVENTION

[0039] HCV replicons and HCV replicon enhanced cells can be used toproduce a cell culture providing detectable levels of HCV RNA and HCVprotein. HCV replicons and HCV replicon enhanced hosts can both beobtained by selecting for the ability to maintain an HCV replicon in acell. As illustrated in the examples provided below, adaptive mutationspresent in HCV replicons and host cells can both assist repliconmaintenance in a cell.

[0040] The detectable replication and expression of HCV RNA in a cellculture system has a variety of different uses including being used tostudy HCV replication and expression, to study HCV and host cellinteractions, to produce HCV RNA, to produce HCV proteins, and toprovide a system for measuring the ability of a compound to modulate oneor more HCV activities.

[0041] Preferred cells for use with a HCV replicon are Huh-7 cells andHuh-7 derived cells. “Huh-7 derived cells” are cell produced startingwith Huh-7 cells and introducing one or more phenotypic and/or genotypicmodifications.

Adaptive Mutations

[0042] Adaptive mutations enhance the ability of an HCV replicon to bcmaintained and expressed in a host cell. Adaptive mutations can beinitially selected for using a wild type HCV RNA construct or a mutatedHCV replicon. Initial selection involves providing HCV replicons tocells and identifying clones containing a replicon.

[0043] Nucleic acid sequences of identified HCV replicons can bedetermined using standard sequencing techniques. Comparing the sequenceof input HCV constructs and selected constructs provides the location ofmutations. The effect of particular mutation(s) can be measured by, forexample, producing a construct to contain particular mutation(s) andmeasuring the effect of these mutation(s). Suitable control constructsfor comparison purposes include wild type constructs and constructspreviously evaluated.

[0044] Adaptive mutations were predominantly found in the HCV NS3 andNS5A regions. With the exception of two silent mutations in NS5A andNS5B, consensus mutations occurring in the NS region resulted in changesto the deduced amino acid sequence. Noticeably, the amino acid changesoccurred in residues that are conserved in all or a large number ofnatural HCV isolates. HCV sequences are well known in the art and can befound, for example, in GenBank.

[0045] Adaptive mutations described herein can be identified withrespect to a reference sequence. The reference sequence provides thelocation of the adaptiv mutation in, for example, the NS3 or NS5A RNA,cDNA, or amino acid sequence. The remainder of the sequence encodes fora functional protein that may have the same, or a different, sequencethan the reference sequence.

[0046] Preferred NS3 and NS5A adaptive mutations and examples of changesthat can be made to produce such mutations are shown in Tables 1 and 2.The amino acid numbering shown in Tables 1 and 2 is with respect to SEQ.ID. NO. 1. The nucleotide numbering shown in Tables 1 and 2 is withrespect to SEQ. ID. NO. 2. SEQ. ID. NO. 1 provides the amino acidsequence of the Con 1 HCV isolate (Accession Number AJ238799). SEQ. ID.NO. 2 provides the nucleic acid sequence of the Con1 HCV isolate. TABLE1 Preferred NS3 Adaptive Mutations Amino Acid Nucleotide gly1095alaG3625C glu1202gly A3946G ala1347thr G4380A

[0047] TABLE 2 Preferred NS5A Adaptive Mutations Amino Acid NucleotideLys@2039 AAA@6458 asn2041thr A6463C ser2173phe C6859T ser2197phe C6931Tleu2198ser T6934C ala2199thr G6936A ser2204arg C6953A (or G)

[0048] Preferred adaptive mutations identified with respect to areference sequence can be produced changing the encoding region of SEQ.ID. NO. 1, or an equivalent sequence, to result in the indicated change.Preferred adaptive mutations provided in Tables 1 and 2 occur in aminoacids conserved among different HCV isolates.

[0049] Adaptive mutations have different effects. Some mutations alone,or in combination with other mutations, enhance HCV replicon activity.In some cases, two or more mutations led to synergistic effects and inone case, a slightly antagonistic effect was observed.

[0050] An adaptive mutation once identified can be introduced into astarting construct using standard genetic techniques. Examples of suchtechniques are provided by Ausubel, Current Protocols in MolecularBiology, John Wiley, 1987-1998, and Sambrook, et al., Molecular Cloning,A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor LaboratoryPress, 1989.

[0051] HCV replicons containing adaptive mutations can be built aroundan NS3 region or NS5A region containing one or more adaptive mutationsdescribed herein. The final replicon will contain replicon componentsneeded for replication and may contain additional components.

[0052] SEQ. ID. NO. 2 can be used as a reference point for different HCVregions as follows:

[0053] 5′ UTR-nucleotides 1-341;

[0054] Core-nucleotides 342-914;

[0055] E1-nucleotides 915-1490;

[0056] E2-nucleotides 1491-2579;

[0057] P7-nucleotides 2580-2768;

[0058] NS2-nucleotides 2769-3419;

[0059] NS3-nucleotides 3420-5312;

[0060] NS4A-nucleotides 5313-5474;

[0061] NS4B-nucleotides 5475-6257;

[0062] NS5A-nucleotides 6258-7598;

[0063] NS5B-nucleotides 7599-9371; and

[0064] 3′ UTR-nucleotides 9374-9605.

[0065] The amino acid sequences of the different structural andnon-structural regions is provided by SEQ. ID. NO. 1.

[0066] Nucleic acid sequences encoding for a particular amino acid canbe produced taking into account the degeneracy of the genetic code. Thedegeneracy of the genetic code arises because almost all amino acids areencoded for by different combinations of nucleotide triplets or“codons”. The translation of a particular codon into a particular aminoacid is well known in the art (see, e.g., Lewin GENES IV, p. 119, OxfordUniversity Press, 1990). Amino acids are encoded for by RNA codons asfollows:

[0067] A=Ala=Alanine: codons GCA, GCC, GCG, GCU

[0068] C=Cys=Cysteine: codons UGC, UGU

[0069] D=Asp=Aspartic acid: codons GAC, GAU

[0070] E=Glu=Glutamic acid: codons GAA, GAG

[0071] F=Phe=Phenylalanine: codons UUC, UUU

[0072] G=Gly=Glycine: codons GGA, GGC, GGG, GGU

[0073] H=His=Histidine: codons CAC, CAU

[0074] I=Ile=Isoleucine: codons AUA, AUC, AUU

[0075] K=Lys=Lysine: codons AAA, AAG

[0076] L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU

[0077] M=Met=Methionine: codon AUG

[0078] N=Asn=Asparagine: codons AAC, AAU

[0079] P=Pro=Proline: codons CCA, CCC, CCG, CCU

[0080] Q=Gln=Glutamine: codons CAA, CAG

[0081] R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU

[0082] S=Set=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU

[0083] T=Thr-Threonine: codons ACA, ACC, ACG, ACU

[0084] V=Val=Valine: codons GUA, GUC, GUG, GUU

[0085] W=Trp=Tryptophan: codon UGG

[0086] Y=Tyr-Tyrosine: codons UAC, UAU.

[0087] Constructs, including subgenomic and genomic replicons,containing one or more of the adaptive mutations described herein canalso contain additional mutations. The additional mutations may beadaptive mutations and mutations not substantially inhibiting repliconactivity. Mutations not substantially inhibiting replicon activityprovide for a replicon that can be introduced into a cell and havedetectable activity.

HCV Replicon

[0088] HCV replicons include the full length HCV genome and subgenomicconstructs. A basic HCV replicon is a subgenomic construct containing anHCV 5′ UTR-PC region, an HCV NS3-NS5B polyprotein encoding region, and aHCV 3′ UTR. Other nucleic acid regions can be present such as thoseproviding for HCV NS2, structural HCV protein(s) and non-HCV sequences.

[0089] The HCV 5′ UTR-PC region provides an internal ribosome entry site(IRES) for protein translation and elements needed for replication. TheHCV 5′ UTR-PC region includes naturally occurring HCV 5′ UTR extendingabout 36 nucleotides into a HCV core encoding region, and functionalderivatives thereof. The 5′-UTR-PC region can be present in differentlocations such as site downstream from a sequence encoding a selectionprotein, a reporter, protein, or an HCV polyprotein.

[0090] Functional derivatives of the 5′-UTR-PC region able to initiatetranslation and assist replication can be designed taking intostructural requirements for HCV translation initiation. (See, forexample, Honda, et al., 1996. Virology 222, 31-42). The affect ofdifferent modifications to a 5′ UTR-PC region can be determined usingtechniques that measure replicon activity.

[0091] In addition to the HCV 5′ UTR-PC region, non-HCV IRES elementscan also be present in the replicon. The non-HCV IRES elements can bepresent in different locations including immediately upstream the regionencoding for an HCV polyprotein. Examples of non-HCV IRES elements thatcan be used are the EMCV IRES, poliovirus IRES, and bovine viraldiarrhea virus IRES.

[0092] The HCV 3′ UTR assists HCV replication. HCV 3′ UTR includesnaturally occurring HCV 3′ UTR and functional derivatives thereof.Naturally occurring 3′ UTR's include a poly U tract and an additionalregion of about 100 nucleotides. (Tanaka, et al., 1996. J. Virol. 70,3307-3312, Kolykhalov, et aL, 1996. J. Virol. 70, 3363-3371.) At leastin vivo, the 3′ UTR appears to be essential for replication.(Kolykhalov, et al., 2000. J. Virol. 2000 4, 2046-2051.) Examples ofnaturally occurring 3′ UTR derivatives are described by BartenschlagerInternational Publication Number EP 1 043 399.

[0093] The NS3-NS5B polyprotein encoding region provides for apolyprotein that can be processed in a cell into different proteins.Suitable NS3-NS5B polyprotein sequences that may be part of a repliconinclude those present in different HCV strains and functionalequivalents thereof resulting in the processing of NS3-NS5B to a producefunctional replication machinery. Proper processing can be measured forby assaying, for example, NS5B RNA dependent RNA polymerase.

[0094] The ability of an NS5B protein to provide RNA polymerase activitycan be measured using techniques well known in the art. (See, forexample, De Franscesco, et al., International Publication Number WO96/37619, Behrens, et al., 1996. EMBO 15:12-22, Lohmann, et al., 1998.Virology 249:108-118.) Preferably, the sequence of the active NS5B issubstantially similar as that provided in SEQ. ID. NO. 1, or a wild typeNS5B such as strains HCV-1, HCV-2, HCV-BK, HCV-J, HCV-N, HCV-H. Asubstantially similar sequence provides detectable HCV polymeraseactivity and contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 amino acid alterations to that present in a HCV NS5B polymerase.Preferably, no more than 1, 2, 3, 4 or 5 alterations are present.

[0095] Alterations to an amino acid sequence provide forsubstitution(s), insertion(s), deletion(s) or a combination thereof.Sites of different alterations can be designed taking into account theamino acid sequences of different NS5B polymerases to identify conservedand variable amino acid, and can be empirically determined.

[0096] HCV replicons can be produced in a wide variety of differentcells and in vitro. Suitable cells allow for the transcription of anucleic acid encoding for an HCV replicon.

Additional Sequences

[0097] An HCV replicon may contain non-HCV sequences in addition to HCVsequences. The additional sequences should not prevent replication andexpression, and preferably serve a useful function. Sequences that canbe used to serve a useful function include a selection sequence, areporter sequence, transcription elements and translation elements.

[0098] Selection Sequence

[0099] A selection sequence in an HCV replicon facilitates theidentification of a cell containing the replicon. Selection sequencesare typically used in conjunction with some selective pressure thatinhibits growth of cells not containing the selection sequence. Examplesof selection sequences include sequences encoding for antibioticresistance and ribozymes.

[0100] Antibiotic resistance can be used in conjunction with anantibiotic to select for cells containing replicons. Examples ofselection sequences providing for antibiotic resistance are sequencesencoding resistance to neomycin, hygromycin, puromycin, or zeocin.

[0101] A ribozyme serving as a selection sequence can be used inconjunction with an inhibitory nucleic acid molecule that preventscellular growth. The ribozyme recognizes and cleaves the inhibitorynucleic acid.

[0102] Reporter Sequence

[0103] A reporter sequence can be used to detect replicon replication orprotein expression. Preferred reporter proteins are enzymatic proteinswhose presence can be detected by measuring product produced by theprotein. Examples of reporter proteins include, luciferase,beta-lactamase, secretory alkaline phosphatase, beta-glucuronidase,green fluorescent protein and its derivatives. In addition, a reporternucleic acid sequence can be used to provide a reference sequence thatcan be targeted by a complementary nucleic acid. Hybridization of thecomplementary nucleic acid to its target can be determined usingstandard techniques.

[0104] Additional Sequence Configuration

[0105] Additional non-HCV sequences are preferable 5′ or 3′ of an HCVreplicon genome or subgenomic genome region. However, the additionalsequences can be located within an HCV genome as long as the sequencesdo not prevent detectable replicon activity. If desired, additionalsequences can be separated from the replicon by using a ribozymerecognition sequence in conjunction with a ribozyme.

[0106] Additional sequences can be part of the same cistron as the HCVpolyprotein or can be a separate cistron. If part of the same cistron,the selection or reporter sequence coding for a protein should result ina product that is either active as a chimeric protein or is cleavedinside a cell so it is separated from HCV protein.

[0107] Selection and reporter sequences encoding for a protein whenpresent as a separate cistron should be associated with elements neededfor translation. Such elements include a 5′ IRES.

Detection Methods

[0108] Methods for detecting replicon activity include those measuringthe production or activity of replicon RNA and encoded for protein.Measuring includes qualitative and quantitative analysis.

[0109] Techniques suitable for measuring RNA production include thosedetecting the presence or activity of RNA. The presence of RNA can bedetected using, for example, complementary hybridization probes orquantitative PCR. Techniques for measuring hybridization betweencomplementary nucleic acid and quantitative PCR are well known in theart. (See for example, Ausubel, Current Protocols in Molecular Biology,John Wiley, 1987-1998, Sambrook, et al., Molecular Cloning, A LaboratoryManual, 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989, andU.S. Pat. No. 5,731,148.)

[0110] RNA enzymatic activity can be provided to the replicon by using aribozyme sequence. Ribozyme activity can be measured using techniquesdetecting the ability of the ribozyme to cleave a target sequence.

[0111] Techniques for measuring protein production include thosedetecting the presence or activity of a produced protein. The presenceof a particular protein can be determined by, for example, immunologicaltechniques. Protein activity can be measured based on the activity of anHCV protein or a reporter protein sequence.

[0112] Techniques for measuring HCV protein activity vary depending uponthe protein that is measured. Techniques for measuring the activity ofdifferent nonstructural proteins such as NS2/3, NS3, and NS5B, are wellknown in the art. (See, for example, references provided in theBackground of the Invention.)

[0113] Assays measuring replicon activity also include those detectingvirion production from a replicon that produces a virion; and thosedetecting a cytopathic effect from a replicon producing proteinsexerting such an effect. Cytopathic effects can be detected by assayssuitable to measure cell viability.

[0114] Assays measuring replicon activity can be used to evaluate theability of a compound to modulate HCV activities. Such assays can becarried out by providing one or more test compounds to a cell expressingan HCV replicon and measuring the effect of the compound on repliconactivity. If a preparation containing more than one compound is found tomodulate replicon activity, individual compounds or smaller groups ofcompounds can be tested to identify replicon active compounds.

[0115] Compounds identified as inhibiting HCV activity can be used toproduce replicon enhanced cells and may be therapeutic compounds. Theability of a compound to serve as a therapeutic compound can beconfirmed using animal models such as a chimpanzee to measure efficacyand toxicity.

Replicon Enhanced Host Cell

[0116] Replicon enhanced cells are initially produced by selecting for acell able to maintain an HCV replicon and then curing the cell of thereplicon. Cells produced in this fashion were found to have an increasedability to maintain a replicon upon subsequent HCV replicontransfection.

[0117] Initial transfection can be performed using a wild-type repliconor a replicon containing one or more adaptive mutations. If a wild-typereplicon is employed, the replicon should contain a selection sequenceto facilitate replicon maintenance.

[0118] Cells can be cured of replicons using different techniques suchas those employing replicon inhibitory agent. In addition, replicationof HCV replicons is substantially reduced in confluent cells. Thus, itis conceivable to cure cells of replicons by culturing them at a highdensity.

[0119] Replicon inhibitory agents inhibit replicon activity or selectagainst a cell containing a replicon. An example of such an agent isIFN-α. Other HCV inhibitory compounds may also be employed. HCVinhibitor compounds are described, for example, in Llinas-Brunet, etal., 2000. Bioorg Med Chem. Lett. 10(20), 2267-2270.

[0120] The ability of a cured cell to be a replicon enhanced cell can bemeasured by introducing a replicon into the cell and determiningefficiency of subsequent replicon maintenance and activity.

EXAMPLES

[0121] Examples are provided below to further illustrate differentfeatures of the present invention. The examples also illustrate usefulmethodology for practicing the invention. These examples do not limitthe claimed invention.

Example 1

[0122] Techniques

[0123] This example illustrates the techniques employed for producingand analyzing adaptive mutations and replicon enhanced cells.

[0124] Manipulation of Nucleic Acids and Construction of RecombinantPlasmids

[0125] Manipulation of nucleic acids was done according to standardprotocols. (Sambrook, et al., 1989. Molecular Cloning: A LaboratoryManual, 2^(nd) ed. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) Plasmid DNA was prepared from ON culture in LB broth using Qiagen500 columns according to manufacturer instructions.

[0126] Plasmids containing desired mutations were constructed byrestriction digestion using restriction sites flanking the mutations orby PCR amplification of the area of interest, using syntheticoligonucleotides with the appropriate sequence. Site directedmutagenesis was carried out by inserting the mutations in the PCRprimers. PCR amplification was performed using high fidelitythermostable polymerases or mixtures of polymerases containing aproofreading enzyme. (Barnes, et al., 1994. Proc. Natl. Acad. Sci. 91,2216-2220.) All plasmids were verified by restriction mapping andsequencing.

[0127] pHCVneo17.wt contains the cDNA for an HCV bicistronic repliconidentical to replicon I₃₇₇neo/NS3-3′/wt described by Bartenschlager(SEQ. ID. NO. 3) (Lohmann, et al., 1999. Science 285,110-113,EMBL-genbank No. AJ242652). The plasmid comprises the followingelements: 5′ untranslated region of HCV comprising the HCV-IRES and partof the core (nt1-377); neomycin phosphotransferase coding sequence; andEMCV IRES; HCV coding sequences from NS3 to NS5B; 3′ UTR of HCV.

[0128] Plasmid pHCVNeo17.GAA is identical to pHCVNeo.17, except that theGAC triplets (nt. 6934-6939 of pHCVNeo17 sequence) coding for thecatalytic aspartates of the NS5B polymerase (amino acids 2737 and 2738of HCV polyprotein) were changed into GCG, coding for alanine.

[0129] Plasmid pHCVNeo17.m0 is identical to pHCVNeo17, except that thetriplet AGC (nt. 5335-5337 of pHCVNeo17 sequence) coding for the serineof NS5A protein (amino acid 2204 of HCV polyprotein) was changed intoAGA, coding for arginine.

[0130] Plasmid pHCVNeo17.m1 is identical to pHCVNeo17, except that thetriplet AAC (nt. 4846-4848 of pHCVNeo17 sequence) coding for theasparagine of NS5A protein (amino acid 2041 of HCV polyprotein) waschanged into ACC, coding for threonine.

[0131] Plasmid pHCVNeo17.m2 is identical to pHCVNeo17, except that thetriplet TCC (nt. 5242-5244 of pHCVNeo17 sequence) coding for the serineof NS5A protein (amino acid 2173 of HCV polyprotein) was changed intoTTC, coding for phenylalanine.

[0132] Plasmid pHCVNeo17.m3 is identical to pHCVNeo17, except that thetriplet TCC (nt. 5314-5316 of pHCVNeo17 sequence) coding for the serineof NS5A protein (amino acid 2197 of HCV polyprotein) was changed intoTTC, coding for phenylalanine.

[0133] Plasmid pHCVNeo17.m4 is identical to pHCVNeo17, except that thetriplet TTG (nt. 5317-5319 of pHCVNeo17 sequence) coding for the leucineof NS5A protein (amino acid 2198 of HCV polyprotein) was changed intoTCG, coding for serine.

[0134] Plasmid pHCVNeo17.m5 is identical to pHCVNeo17, except that anextra triplet AAA coding for lysine was inserted after the triplet GTG(nt. 4840-4843 of pHCVNeo17 sequence), coding for valine 2039 of HCVpolyprotein. Plasmid pHCVNeo17.m6 is identical to pHCVNeo17, except thatthe triplets GAA and GCC (nt. 2329-2331 and 2764-2766 of pHCVNeo17sequence) coding for the glutamic acid and the alanine of NS3 protein(amino acid 1202 and 1347 of HCV polyprotein) were changed respectivelyinto GGA and ACC, coding for glycine and threonine. The triplet TCC (nt.5242-5244 of pHCVNeo17 sequence) coding for the serine of NS5A protein(amino acid 2173 of HCV polyprotein) was changed into TTC, coding forphenylalanine; an extra adenosine was inserted into the EMCV TRES (afterthe thymidine 1736 of the replicon sequence).

[0135] Plasmid pHCVNeo17.m7 is identical to pHCVNeo17, except that thetriplet AAC (nt. 4846-4848 of pHCVNeo17 sequence) coding for theasparagine of NS5A protein (amino acid 2041 of HCV polyprotein) waschanged into ACC, coding for threonine; the triplet TCC (nt. 5242-5244of pHCVNeo17 sequence) coding for the serine of NS5A protein (amino acid2173 of HCV polyprotein) was changed into TTC, coding for phenylalanine.

[0136] Plasmid pHCVNeo17.m8 is identical to pHCVNeo17, except that thetriplet AAC (nt. 4846-4848 of pHCVNeo17 sequence) coding for theasparagine of NS5A protein (amino acid 2041 of HCV polyprotein) waschanged into ACC, coding for threonine; the triplet TCC (nt. 5314-5316of pHCVNeo 17 sequence) coding for the serine of NS5A protein (aminoacid 2197 of HCV polyprotein) was changed into TTC, coding forphenylalanine.

[0137] Plasmid pHCVNeo17.m9 is identical to pHCVNeo17, except that thetriplet AAC (nt. 4846-4848 of pHCVNeo17 sequence) coding for theasparagine of NS5A protein (amino acid 2041 of HCV polyprotein) waschanged into ACC, coding for threonine; the triplet TTG (nt. 5317-5319of pHCVNeo17 sequence) coding for the leucine of NS5A protein (aminoacid 2198 of HCV polyprotein) was changed into TCG, coding for serine.

[0138] Plasmid pHCVNeo17.m10 is identical to pHCVNeo17, except that thetriplet GAA (nt. 2329-2331 of pHCVNeo17 sequence) coding for theglutamic acid of NS3 protein (amino acid 1202 of HCV polyprotein) waschanged into GGA, coding for glycine; an extra triplet AAA coding forlysine was inserted after the triplet GTG (nt. 4840-4843 of pHCVNeo17sequence), coding for valine 2039 of HCV polyprotein.

[0139] Plasmid pHCVNeo17.m11 is identical to pHCVNeo17, except that thetriplet TCC (nt. 5314-5316 of pHCVNeo17 sequence) coding for the serineof NS5A protein (amino acid 2197 of HCV polyprotein) was changed intoTTC, coding for phenylalanine. The triplet GCC (nt. 5320-5322 ofpHCVNeo17 sequence) coding for the alanine of NS5A protein (amino acid2199 of HCV polyprotein) was changed into ACC coding for threonine.

[0140] Plasmid pHCVNeo17.m12 is identical to pHCVNeo17, except that thetriplet AAC (nt. 4846-4848 of pHCVNeo17 sequence) coding for theasparagine of NS5A protein (amino acid 2041 of HCV polyprotein) waschanged into ACC, coding for threonine; the triplet TCC (nt. 5314-5316of pHCVNeo17 sequence) coding for the serine of NS5A protein (amino acid2197 of HCV polyprotein) was changed into TTC, coding for phenylalanine.The triplet GCC (nt. 5320-5322 of pHCVNeo17 sequence) coding for thealanine of NS5A protein (amino acid 2199 of HCV polyprotein) was changedinto ACC coding for threonine.

[0141] Plasmid pHCVNeo17.m13 has the same mutations as pHCVNeo17.m8, butalso an extra adenosine inserted into the EMCV IRES (after the thymidine1736 of the replicon sequence).

[0142] Plasmid pHCVNeo17.m14 has the same mutations as pHCVNeo17.m11,but also an extra adenosine inserted into the EMCV IRES (after thethymidine 1736 of the replicon sequence).

[0143] Plasmid pHCVNeo17.m15 is identical to pHCVNeo17, except that thetriplet GCC (nt. 5320-5322 of pHCVNeo17 sequence) coding for the alanineof NS5A protein (amino acid 2199 of HCV polyprotein) was changed intoACC coding for threonine.

[0144] Plasmid pRBSEAP.5 is a pHCVNeo.17 derivative where the Neo codingsequence has been replaced with the sequence coding for the humanplacental alkaline phosphatase corresponding to nucleotides 90-1580 ofpBC12/RSV/SEAP plasmid. (Berger, et al., 1988. Gene 66, 1-10.)

[0145] RNA Transfection

[0146] Transfection was performed using Huh-7 cells. The cells weregrown in Dulbecco's modified minimal essential medium (DMEM, Gibco, BRL)supplemented with 10% FCS. For routine work, cells were passed 1 to 5twice a week using 1× trypsin/EDTA (Gibco, BRL).

[0147] Plasmids were digested with the Scal endonuclease (New EnglandBiolabs) and transcribed in vitro with the T7 Megascript kit (Ambion).Transcription mixtures were treated with DNase I(0.1 U/ml) for 30minutes at 37° C. to completely remove template DNA, extracted accordingto the procedure of Chomczynski (Chomczynski, et al., 1987. Anal.Biochem. 162, 156-159), and resuspended with RNase-free phosphatebuffered saline (rfPBS, Sambrook, et al., 1989. Molecular Clotting: ALaboratory Manual, 2^(nd) ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

[0148] RNA transfection was performed as described by Liljestrom, etal., 1991. J. Virol. 6, 4107-4113, with minor modifications.Subconfluent, actively growing cells were detached from the tissueculture container using trypsin/EDTA. Trypsin was neutralised byaddition of 3 to 10 volumes of DMEM/10% FCS and cells were centrifugedfor 5 minutes at 1200 rpm in a Haereus table top centrifuge at 4° C.Cells were resuspended with ice cold rfPBS by gentle pipetting, countedwith a haemocitometer, and centrifuged as above. rfPBS wash was repeatedonce and cells were resuspended at a concentration of 1-2×10⁷ cell/ml inrfPBS. Aliquots of cell suspension were mixed with RNA in sterileeppendorf tubes. The RNA/cell mixture was immediately transferred intothe electroporation cuvette (precooled on ice) and pulsed twice with agene pulser apparatus equipped with pulse controller (Biorad). Dependingon the experiment, 0.1, 0.2 or 0.4 cm electrode gap cuvettes were used,and settings adjusted (Table 3). TABLE 3 Cuvette Volume VoltageCapacitance Resistance RNA gap (cm) (μl) (Volts) (μFa) (ohm) (μg) 0.1 70 200 25 infinite 1-10 0.2 200 400 25 infinite 5-20 0.4 800 800 25infinite 15-100

[0149] After the electric shock, cells were left at room temperature for1-10 minutes (essentially the time required to electroporate allsamples) and subsequently diluted with at least 20 volumes of DMEM/10%FCS and plated as required for the experiment. Survival and transfectionefficiency were monitored by measuring the neutral red uptake of cellcultured for various days in the absence or in the presence of neomycinsulfate (G418). With these parameters, survival of Huh-7 cells wasusually 40-60% and transfection efficiency ranged between 40% and 100%.

[0150] Sequence Analysis of Replicon RNAs

[0151] The entire NS region was recloned from 3 different transfectionexperiments performed with HCVNeo.17 RNA. RNA was extracted fromselected clones either using the Qiagen RNAeasy minikit followingmanufacturer instructions or as described by Chomczynski, et al., 1987.Anal. Biochem. 162, 156-159.

[0152] Replicon RNAs (5 μg of total cellular RNA) were retro-transcribedusing oligonucleotide HCVG34 (5′-ACATGATCTGCAGAGAGGCCAGT-3′; SEQ. ID.No. 4) and the Superscript II reverse transcriptase (Gibco, BRL)according to manufacturer instructions, and subsequently digested with 2U/ml Ribonuclease H (Gibco BRL). The cDNA regions spanning from the EMCVIRES to the HCV 3′ end were amplified by PCR using oligonucleotidesHCVG39 (5′-GACASGCTGTGATAWATGTCTCCCCC-3′; SEQ. ID. NO. 5) and CITE3(5′-TGGCTCTCCTCAAGCGTATTC-3′; SEQ. ID. NO. 6) and the LA Taq DNApolymerase (Takara LA Taq).

[0153] Amplified cDNAs were digested with the KpnI end endouclease (NewEngland Biolabs) and the 5.8 kb fragments were gel purified and ligatedto the 5.6 kb vector fragment (purified from plasmid pRBSEAP.5 digestedwith Kpnl) using T4 DNA ligase (New England Biolabs) according tomanufacturer instructions. Ligated DNAs were transformed byelectroporation in DH10B or JM119 strains of E. coli.

[0154] In the case of NS5A region, total RNA isolated from 3 clones,(HB77, HB60 and HB68) was extracted and used for RT-PCR. 5 μg of totalRNA plus 20 pmole of AS61 oligo (5′-ACTCTCTGCAGTCAAGCGGCTCA-3′, RTantisense oligo; SEQ. ID. NO. 7) were heated 5 minutes at 95° C., thenDMSO (5% f.c.), DTT (10 mM f.c.), 1 mM dNTP (1 mM f.c.), 1× Superscriptbuffer (1×f.c.), and 10 μSuperscript (Gibco) were added to a totalvolume of 20 μl and incubated 3 hours at 42° C. 2 μl of this RT reactionwere used to perform PCR with oligos S39 (5′-CAGTGGATGAACCGGCTGATA-3′,sense; SEQ. ID. NO. 8) or S41 (5′-GGGGCGACGGCATCATGCAAACC-3′, sense;SEQ. ID. NO. 9) and B43 (5′-CAGGACCTGCAGTCTGTCAAAGG-3′, antisense; SEQ.ID. NO. 10) using Elongase Enzyme Mix (Gibco) according the instructionprovided by the manufacturer. The resulting PCR fragment was cloned inpCR2.1 vector using the TA Cloning kit (Invitrogen) and transformed inTop10F′ bacterial strain.

[0155] Plasmid DNA was prepared from ON culture of the resultingampicillin resistant colonies using Qiagen 500 columns according tomanufacturer instructions. The presence of the desired DNA insert wasascertained by restriction digestion, and the nucleotide sequence ofeach plasmid was determined by automated sequencing. Nucleotidesequences and deduced amino acids sequences were aligned using the GCGsoftware.

[0156] TaqMan

[0157] TaqMan analysis was typically performed using 10 ng of RNA in areaction mix (TaqMan Gold RT-PCR kit, Perkin Elmer Biosystems) eitherwith HCV specific oligos/probe (oligo 1: 5′-CGGGAGAGCCATAGTGG-3′; SEQ.ID. NO. 11, oligo 2: 5′-AGTACCACAAGGCCTITCG-3′; SEQ. ID. NO. 12, probe:5′-CTGCGGAACCGGTGAGTACAC-3′; SEQ. ID. NO. 13) or with human GAPDHspecific oligos/probe (Pre-Developed TaqMan Assay Reagents, EndogenousControl Human GAPDH, Part Number 4310884E, Perkin Elmer Biosystems). PCRwas performed using a Perkin Elmer ABI PRISM 7700 under the followingconditions: 30 minutes at 48° C. (the RT step), 10 minutes at 95° C. and40 cycles: 15 seconds at 95° C. and 1 minute at 60° C. Quantitativecalculations were obtained using the Comparative C_(T) Method (describedin User Bulletin #2, ABI PRISM 7700 Sequence Detection System, AppliedBiosystem, December 1997) considering the level of GAPDH mRNA constant.All calculations of HCV RNA are expressed as fold difference over aspecific control.

[0158] Antibodies and Immunological Techniques

[0159] Mouse monoclonal antibody (anti-NS3 mab10E5/24) were produced bystandard techniques. (Galfré and Milstein, 1981. Methods in Enzymology73, 1-46.) Purified recombinant protein was used as an immunogen.(Gallinari, et al., 1999. Biochemistry 38, 5620-5632.)

[0160] For Cell-ELISA analysis, transfected cells were monitored forexpression of the NS3 protein by ELISA with the anti-NS3 mab 10E5/24.Cells were seeded into 96 well plates at densities of 40,000, 30,000,15,000 and 10,000 cells per well and fixed with ice-cold isopropanol at1, 2, 3 and 4 days post-transfection, respectively. The cells werewashed twice with PBS, blocked with 5% non-fat dry milk in PBS +0.1%Triton X100 +0.02% SDS (PBSTS) and then incubated overnight at 4° C.with 10E5/24 mab diluted 1:2000 in Milk/PBSTS. After washing 5 timeswith PBSTS, the cells were incubated for 3 hours at room temperaturewith anti-mouse IgG Fc specific alkaline phosphatase conjugatedsecondary antibody (Sigma A-7434), diluted 1:2000 in Milk/PBSTS. Afterwashing again as above, the reaction was developed with p-nitrophenylphosphate disodium substrate (Sigma 104-105) and the absorbance at 405nm read at intervals.

[0161] The results were normalized by staining with sulforhodamine B(SRB Sigma S 1402) to determine cell numbers. The alkaline phosphatasesubstrate was removed from the wells and the cells washed with PBS. Theplates were then incubated with 0.4% SRB in 1% acetic acid for 30minutes (200 μl/well), rinsed 4 times in 1% acetic acid, blotted dry andthen 200 μl/well of 10 mM Tris pH 10.5 added. After mixing, theabsorbance at 570 nm was read.

[0162] Neutral Red/Crystal Violet Staining of Foci

[0163] The survival of transfected cells in the absence or presence ofG418 was monitored by staining of foci/clones with neutral red in vivowith subsequent crystal violet staining. The medium was removed from thecells and replaced with fresh medium containing 0.0025% neutral red(Sigma N2889) and the cells incubated for 3 hours at 37° C. Cells werewashed twice with PBS, fixed in 3.5% formaldehyde for 15 minutes, washedtwice again in PBS and then with distilled water and the number of(live) foci counted. The cells could then be re-stained with crystalviolet by incubating with an 0.1% crystal violet (Sigma C0775) solutionin 20% methanol for 20 minutes at room temperature, followed by 3 washesin 20% methanol and a wash with distilled water.

[0164] Preparation of Cells Cured of Endogenous Replicon

[0165] Replicon enhanced cells designated 10IFN and C1.60/cu wereproduced using different HCV inhibitory agents. Based on the techniquesdescribed herein additional replicon enhanced clones can readily beobtained.

[0166] 10IFN was obtained by curing a Huh-7 cell of a replicon usinghuman IFN-α2b. Huh-7 cells containing HCV replicons (designated HBI10,HBIII4, HBIII27 and HBIII18) were cultured for 11 days in the presenceof 100 U/ml recombinant human IFN-α2b (Intron-A, Schering-Plough), andsubsequently for 4 days in the absence of IFN-α2b. At several timepoints during this period, the clones were analyzed for the presence ofHCV proteins and RNA by Western and Northern blotting. After 7 days ofincubation with IFN-α2b, HCV proteins could no longer be detected in anyof these clones by Western blotting and similar effects were seen withRNA signals in Northern blots. IFN-α2b treated cells were stored inliquid nitrogen until used for transfection experiments.

[0167] C1.60/cu was obtained by curing a Huh-7 cell of a replicon usingan HCV inhibitory compound. The presence of HCV RNA was determined usingPCR (TaqMan) at 4, 9, 12 and 15 days. From day 9 the amount of HCV RNAwas below the limit of detection. To further test the disappearance ofthe replicon, 4 million cells of cured Clone 60 cells (after the 15 daysof treatment) were plated in the presence of 1 mg/ml G418. No viablecells were observed, confirming that absence of HCV replicons able toconfer G418 resistance.

Example 2

[0168] Selection and Characterization of Cell Clones ContainingFunctional HCV Replicons

[0169] Huh-7 cells (2-8×10⁶) were transfected by electroporation with invitro transcribed replicon RNAs (10-20 μg), plated at a density rangingfrom 2.5×10³ to 10×10³/cm², and cultured in the presence of 0.8-1 mg/mlG418. The majority of replicon transfected cells became transientlyresistant to G418 and duplicated normally for 7 to 12 days in thepresence of the drug, while cells transfected with irrelevant RNAs andmock transfected cells did not survive more than 7 days (data notshown). Transient resistance to G418 was likely due to persistence ofthe Neo protein expressed from the transfected RNA, since it wasobserved also with mutated replicons unable to replicate. Approximately2 weeks after transfection, transient resistance declined, most cellsdied and small colonies of cells permanently resistant to the antibioticbecame visible in samples transfected with HCVNeo.17 RNA, but not incells transfected with other replicon RNAs.

[0170] In several experiments, the frequency of G418 resistant clonesranged between 10 and 100 clones per 10⁶ transfected cells. About 20G418 resistant colonies were isolated, expanded and molecularlycharacterized. PCR and RT-PCR analysis of nucleic acids indicated thatall clones contained HCV RNA but not HCV DNA, demonstrating that G418resistance was due to the presence of functional replicons (data notshown). This result was confirmed by Northern blot analysis andmetabolic labeling with 3H-uridine, which revealed the presence of bothgenomic and antigenomic HCV RNAs of the expected size (data not shown).Lastly, western blot, immunoprecipitation and immunofluorescenceexperiments showed that these clones expressed all HCV non-structuralproteins as well as Neo protein (data not shown).

[0171] Clones differed in terms of cell morphology and growth rate.Replicon RNA copy number (500-10000 molecules/cell) and viral proteinexpression also varied between different clones (data not shown).However, the amount of replicon RNA and proteins also varied withpassages and with culture conditions and was higher when cells were notallowed to reach confluency, suggesting that replicons replicated moreefficiently in dividing cells than in resting cells. Processing of theviral polyprotein occurred with kinetics similar to those observed intransfected cells.

[0172] Interestingly, in all tested clones HCV replication wasefficiently inhibited by treating the cells with IFN-α2b. The EC₅₀ wasbetween 1 and 10 U/ml, depending on the clone.

Example 3

[0173] Identification of Adaptive Mutations

[0174] The low number of G418 resistant clones derived from HCVNeo.17RNA transfection suggested that replication could require mutation(s)capable of adapting the replicon to the host cell (adaptive mutations)and/or that only a small percentage of Huh-7 cells were competent forHCV replication. To verify the first hypothesis, mutations in repliconsRNAs derived from selected cell clones were identified.

[0175] RNA sequences for different replicons were determined usingstandard techniques. Such techniques involved isolating RNA from severalindependent clones, reverse transcription to produce cDNA, amplifyingcDNAs by PCR and cloning into an appropriate vector. The cDNA spanningalmost the entire HCV NS region (126 bp at the 3′ end of the EMCV IRESand 5650 bp of the HCV NS region (i.e., the entire NS ORF and 298nucleotides at the 3′ end) from 5 clones (HBI10, HBIII12, HBIII8,HBIII27, HBIV1) were recloned and sequenced. In addition, the NS5Acoding region (nt. 4784-6162) from 3 additional clones (HB 77, HB 68 andHB 60) were recloned and sequenced.

[0176] To discriminate mutations present in the replicon RNA from thosederived from the cloning procedure, at least 2 isolates derived fromindependent RT-PCR experiments were sequenced for each cell clone.Comparison of the nucleotide sequences with the parental sequenceindicated that each isolate contained several mutations (Tables 4A and4B). TABLE 4A HBIII 12 HBIII 18 HBI 10 HBIII 27 4 29 28 61 12 43 13 72Cell clone 1674- 1674- 1674- 1674- 1674- 1674- isolate 7460 7460 74607460 7460 7460 1674-7460 1674-7460 EMCV A @ 1736 A @ 1736 C 1752 T T1678 C IRES 126 bp NS3 G 2009 C A 2330 G T 2150 C T 2015 C T 1811 A A2330 G G 2009 C G 2009 C 1895 bp A 2698 G C 2505 T C 2196 A A 2338 G A2330 G A 2882 G T 2015 C C 2052 A G 2764 A G 2764 A T 3023 A C 2616 T T2666 C T 3673 C C 2336 G G 2644 A A 3256 G T 3085 C T 3134 C A 2664 G T3395 C A 3130 T C 2803 A T 3273 C C 3267 T A 3148 G A 3401 G T 2823 A T3286 C A 3518 C T 3692 C C 3615 T C 3657 T NS4A T 3790 C A 3847 G T 3827A T 3742 C A 3743 G A 3797 G 161 bp NS4B T 3869 C C 4283 T G 4300 A A4136 G T 4290 C A 4053 G G 3880 A C 4547T 782 bp A 4107 G C 4429 T A4261 G A 2496 C T 4200 C T 4185 C G 4309 A T 4316 G A 4366 G A 4428 G A4449 G NS5A A 4847 C G 4728 A C 5243 T C 4729 A A 4694 T A 4675 G A 4855G A 4888 G 1340 bp G 5158 A A 4845 G A 5486 G T 4993 C AAA @ A 4761 G C5006 T C 4985 T 4842 G 5175 C C 5243 T C 5596 T G 5095 A T 5237 C AAA @T 5318 C T 5030 A 4842 C 5243 T G 5512 T G 5823 A T 5334 C T 5368 C

T 5090 A C 5390 T A 5521 G A 5374 T G 5866 A T 5318 C A 5719 G A 5600 GT 5379 A A 5328 G A 5740 C T 5480 C A 5399 G A 5513 G A 5574 G T 5977 CNS5B T 6316 C A 6406 G T 6074 C A 6150 G A 6911 G A 5986 G G 6479 C G6156 A 1477 bp T 6589 C G 6756 A A 6541 G A 6218 G T 6099 C C 6870 T G7434 A T 7370 C G 6963 T A 6732 G T 7352 A C 6141 T A 7213 G T 7444 C A7350 T G 6463 A T 7448 C A 7359 G C 6849 T T 6865 C

[0177] TABLE 4B Cell clone HBIV 1 HB 77 HB 68 HB 60 isolate 85 93 10 1442 1 13 7 1674-7460 1674-7460 4784-6162 4465-6162 4784-6162 4465-61624784-6162 4784-6162 EMCV A @ 1736 IRES 126 bp NS3 A 3403 G A 2572 G 1895bp A 3454 G NS4A 161 bp NS4B A 4084 G C 3892 T 782 bp NS5A T 4742 C A4847 C C 4813 T A 4699 C T 5171 G T 4587 C A 4821 G C 5337 G 1340 bp C5315 T A 5225 G G 5060 C A 5161 G C 5298 T T 4972 C G 5320 A C 5551 T G5431 T C 5315 T C 5337 A C 5337 A C 5337 A A 5094 G A 5414 G G 5806 A T5751 C G 5320 A A 5459 G A 5639 G A 5278 G T 5601 G T 5797 C T 5356 A T5977 C A 5969 G G 5320 A C 5808 T G 5523 A C 5532 T T 5888 A NS5B T 6144A T 6855 C 1477 bp A 6365 G A 7135 G A 6656 G T 7171 C A 6677 G T 6855 CT 6947 A T 6997 C G 7041 T A 7187 C

[0178] The frequency of mutations ranged between 1.7×10⁻³ and 4.5×10⁻³(average 3×10⁻³). The majority of mutations were nucleotidesubstitutions, although insertions of 1 or more nucleotides were alsoobserved (Tables 4A and 4B).

[0179] Approximately 85% of the mutations found only in 1 isolate(non-consensus) were randomly distributed in the recloned fragment, andpossibly include mis-incorporation during the PCR amplifications.Conversely, the remaining 15% of the mutations were common to 2 or moreisolates derived from independent RT-PCR experiments (consensusmutations), and presumably reflected mutations present in the templateRNA.

[0180] Consensus mutations were found in all isolates and were eithercommon to isolates derived from the same clone (consensus A), or toisolates derived from different clones (consensus B). Analysis ofadditional isolates derived from the same cell clones indicated thatconsensus A mutations were not always present in all isolates derivedfrom one clone (data not shown). This observation, together with thepresence of consensus B mutations, suggests that, even within a singlecell clone, replicons exist as quasi-species of molecules with differentsequences.

[0181] At variance with non-consensus mutations, consensus mutationswere not randomly distributed but were clustered in the regions codingfor the NS5A protein (frequency 1×10⁻³) and for the NS3 protein(frequency 0.5×10⁻³). Only one consensus mutation was found in theregion coding for the NS5B protein (frequency 0.1×10⁻³ nucleotides) andnone in the regions coding for NS4A and NS4B. Interestingly, 1 consensusmutation was observed also in the EMCV IRES.

[0182] With the exception of 2 silent mutations found in NS5A and NS5B,consensus mutations occurring in the NS region resulted in changes inthe deduced amino acid sequence (Tables 5A and 5B). Noticeably, theseamino acid changes occurred in residues that are conserved in all ormost natural HCV isolates. Interestingly, clones HB 77 and HB 60displayed different nucleotide substitutions (C5337A and C5337G,respectively) resulting in the same amino acidic mutation (S 2204 R).TABLE 5A Cell clone HBIII 12 HBLII 18 HBI 10 HBIII 27 isolate 4 29 28 6112 43 13 72 NS3 G 1095 A E 1202 G E 1202 G E 1202 G G 1095 A G 1095 A A1347 T A 1347 T NS4A NS4B NS5A N 2041 T S 2173 F S 2173 F E 2263 K @2039 K @ 2039 L 2198 S L 2198 S S 2173 F R 2283 R R 2283 R NS5B

[0183] TABLE 5B Cell clone HBIV 1 HB 77 HB 68 HB 60 isolate 85 93 10 1442 1 13 7 NS3 NS4A NS4B NS5A S 2197 F N 2041 T S 2204 R S 2204 R S 2204R A 2199 T A 2199 T S 2204 R S 2197 F A 2199 T NS5B N 2710 N N 2710 N

Example 4

[0184] Functional Characterization of Consensus Mutations

[0185] The identification of consensus mutations in recloned repliconsindicated that replication proficiency of replicon RNAs contained inselected cell clones depended from the presence of such mutations. Tosubstantiate this hypothesis, the effect of several consensus mutationson replication were analyzed.

[0186] Consensus mutations found in the NS5A region were more closelyanalyzed. Consensus mutations were segregated from the non-consensusones, and pHCVNeo.17 derivatives containing single or multiple consensusmutations were constructed (Table 6). TABLE 6 G418 Consensus mutationscfu/10⁵ EMCV transfected Construct NS3 NS5A IRES cells pHCVNeo17.wt 0-3pHCVNeo17.GAA 0 pHCVNeo17.m0 S2204R  30-130 pHCVNeo17.m1 N2041T 0-3pHCVNeo17.m2 S2173F 15-60 pHCVNeo17.m3 S2197F 160-500 pHCVNeo17.m4L2198S 30-50 pHCVNeo17.m5 K@2039 25-55 pHCVNeo17.m6 E1202G; S2173F ExtraA  13-100 A1347T pHCVNeo17.m7 N2041T; S2173F 0-1 pHCVNeo17.m8 N2041T;S2197F 360-500 pHCVNeo17.m9 N2041T; L2198S 140-170 pHCVNeo17.m10 E1202GK@2039 1060 pHCVNeo17.m11 S2197F; A2199T  900 pHCVNeo17.m12 N2041T;S2197F; >1000  A2199T pHCVNeo17.m13 N2041T; S2197F Extra A  100pHCVNeo17.m14 S2197F; A2199T Extra A >500 pHCVNeo17.m15 A2199T 300-600

[0187] RNAs transcribed in vitro from these constructs were transfectedin Huh-7 cells and the affect on replication was estimated by countingneomycin resistant colonies (G418 cfu). As shown in Table 6, all but 1construct containing single consensus mutations showed a significantincrease on G418 cfu efficiency, thus indicating that the correspondingmutations improved replication. Noticeably, 2 mutants containing singlemutations in NS5A (m3 and m15) were clearly more effective than allother single mutants. Results of mutants containing 2 or more mutations,indicated the presence of a synergistic effect in some combinations (m8,m9, m11 and possibly m10), but also a slightly antagonistic effect in 1mutant (m7).

Example 5

[0188] Replicon Replication in the Absence of Selection

[0189] Replication of HCV replicons in the absence of a G418 selectionwas detected using quantitative PCR (TaqMan). At 24 hourspost-transfection a large amount of replicon RNA was detected in cellstransfected with all replicons, including the GAA control repliconcontaining mutations in the catalytic GDD motif of the NS5B polymerase.This result suggested that analysis at very early time points (up to 48hour post-transfection) essentially measured the input RNA. Northernblot analysis also indicated that after 24 hours the majority of thetransfected RNA was degraded intracellularly (data not shown).

[0190] Analysis at later time points showed that the amount of repliconRNA was considerably reduced at 4 days and eventually becameundetectable (6/8 days) in cells transfected with replicon HCVNeo17.wt,but was still high in cells transfected with replicons m0, m3 and m15(Table 7). At day six, that the amount of replicon RNA becameundetectable in cells transfected with replicon HCVNeo17.wt, m0, and m2,but was detectable in cells transfected with replicon m3 and m15 (Table7). TABLE 7 Hu H7 RNA equ. RNA equ. Name day 4 day 6 Wt 1 x 1 xhcvneo17.m0 3 x 1 x hcvneo17.m2 1 x 1 x hcvneo17.m3 5 x 3 x hcvneo17.m156 x 5 x

[0191] Persistence of m0, m3 and m15 replicons RNA was abolished bytreatment with interferon-α or with an HCV inhibitory compound (data notshown). Moreover, RNA persistence was not observed with mutatedreplicons carrying the NS5B GAA mutation besides adaptive mutations(data not shown). Taken together, these results demonstrated thatquantitative PCR could be used to monitor replication at early timespost-transfection, and can be used to evaluate the replicationproficiency of replicon RNAs containing mutations.

[0192] Comparison of the results shown in Tables 6 and 7, indicated thatthere was a good correlation between the amount of replicon RNA detectedby TaqMan and the G418 cfu efficiency. Nonetheless, some mutants (m2,m3) showed a pronounced effect on G418 cfu efficiency, and little if anyeffect on early replication as measured by TaqMan PCR, while othermutants (m0) showed the reverse behavior.

Example 6

[0193] HCV Replicon Enhanced Cells

[0194] HCV replicon enhanced cells were produced by introducing an HCVreplicon into a host, then curing the host of the replicon. Adaptivemutations (or combinations of them) by themselves increased up to 2orders of magnitude the G418 cfu efficiency and enhanced earlyreplication comparably. Nonetheless, even with the most effectivemutants, only a small percentage of transfected cells (<5%, data notshown) gave rise to G418 resistant clones containing functionalreplicons. This observation was attributed, at least in part to a lowcloning efficiency of Huh-7 cells (data not shown), and only a fractionof Huh-7 cells being competent for replication.

[0195] Several clones were cured of endogenous replicons by treatingthem for about 2 weeks with IFN-α or with a HCV inhibitory compound.Analysis at the end of the treatment showed that neither viral proteinsnor replicon RNA could be detected.

[0196] Cured cells (10IFN and Cl.60/cu) were transfected with mutatedreplicons and replication efficiency was determined by counting neomycinresistant clones (10IFN) or by TaqMan (10IFN and Cl.60/cu). As shown inTable 8, for all tested replicons the G418 cfu efficiency in 10IFN cellswas at least 5 fold higher than in parental Huh-7 cells. This increasein G418 cfu efficiency was particularly relevant for a subset of mutants(m3, m5, m8, m9, m15). TABLE 8 Consensus mutations G418 cfu/10⁵ EMCVtransfected Construct NS3 NS5A IRES cells pHCVNeo17.wt 12-56pHCVNeo17.GAA   0 pHCVNeo17.m0 S2204R  180-1000 pHCVNeo17.m1 N2041T 8-13 pHCVNeo17.m2 S2173F  2000 pHCVNeo17.m3 S2197F 1600-3000pHCVNeo17.m4 L2198S 190-650 pHCVNeo17.m5 K@2039 1600-3000 pHCVNeo17.m6E1202G; S2173F extra A  600-2000 A1347T pHCVNeo17.m7 N2041T; 170-800S2173F pHCVNeo17.m8 N2041T; >4000 S2197F pHCVNeo17.m9 N2041T; 1400-3000L2198S pHCVNeo17.m10 E1202G K@2039 >4000 pHCVNeo17.m11 S2197F; >4000A2199T pHCVNeo17.m12 N2041T; >4000 S2197F; A2199T pHCVNeo17.m13 N2041T;extra A >4000 S2197F pHCVNeo17.m14 S2197F; extra A >4000 A2199TpHCVNeo17.m15 A2199T >4000

[0197] Strikingly, the best mutants yielded a number of G418 resistantclones ranging between 20 and 80% of the cell clones which grew in theabsence of G418 (data not shown), thus indicating that the majority of10IFN cells were competent for replication. This result was confirmed byTaqMan analysis (Table 9), in which the fold increase versus theparental Huh-7 cells was very high. The data indicates that repliconscarrying adaptive mutations replicate vigorously in replicon enhancedcells such as 10IFN and C1.60/cu. TABLE 9 10IFN C1.60/cu. RNA equ. RNAequ. RNA equ. RNA equ. Name Day 4 day 6 day 4 Day 6 Wt  1 x  1 x  1 x  1x hcvneo17.m0 46 x 12 x 78 x 512 x hcvneo17.m2  2 x  2 x  1 x  2 xhcvneo17.m3 68 x 49 x 19 x 392 x hcvneo17.m15 247 x  80 x 268 x  5518 x 

[0198] Expression of viral proteins was determined in replicon enhancedcells using an ELISA assay designed to detect the NS3 protein intransfected cells plated in 96 wells microtiter plates (Cell-ELISA). Asshown in Table 10, 24 hours post-transfection cells transfected with alltested replicons expressed low but detectable levels of the NS3 protein.TABLE 10 NS3 arbitrary units Name h p.t. 96 h p.t. Construct − +IFN −+IFN Mock 1 1 1 1 pHCVNeo17.wt 3.7 4.2 1.2 1.3 pHCVNeo17.GAA 3.1 3.2 1.11 pHCVNeo17.m0 3.4 3.2 9.9 0.8 pHCVNeo17.m3 5.7 4.6 4.7 1.5 pHCVNeo17.m86.6 5.1 15.1 1.4 pHCVNeo17.m10 8 5.6 9.2 1.8 pHCVNeo17.m11 8.4 6.2 13.61.8

[0199] The early expression shown in Table 10 is likely due totranslation of transfected RNA, since it was comparable in all replicons(including that carrying the GAA mutation) and was not affected byIFN-α. At 4 days post-transfection, NS3 expression persisted orincreased in cells transfected with replicons carrying consensusmutations, but could not be detected anymore in cells transfected withwt and GAA replicons. In addition, NS3 expression was almost completelyabolished when cells were cultured in the presence of IFN-α.

[0200] Taken together, these results indicated that the level of NS3expression reflected the replication rate. Indeed, NS3 expression level(Table 10) paralleled the RNA level measured by TaqMan (Table 9). Thehigh replication proficiency of 10IFN cells was further confirmed byimmunofluorescence experiments which showed that more than 50% of cellstransfected with replicons m8 and m11 expressed high level of viralproteins, and that expression was almost completely abolished by IFN-α.

Example 7

[0201] Replication of Full Length Constructs

[0202] This example illustrates the ability of a full length HCV genomecontaining adaptive mutations described herein to replicate in areplicon enhanced host cell. The full length sequence of the HCV isolateCon-1 (EMBL-Genbank No. AJ238799) (plasmid pHCVRBFL.wt) and 2derivatives containing either the N2041T and S2173 F mutations (plasmidpHCVRBFL.m8) or the S2197F and A2199T mutations (plasmid pHCVRBFL.m11)were used as starting constructs.

[0203] RNAs transcribed from the starting constructs were transfected in10IFN cells and their replication proficiency was assessed byCell-ELISA, immunofluorescence and TaqMan. Both constructs containingconsensus mutations (pHCVRBFL.m8 and pHCVRBFL.m11) replicated, while nosign of replication was observed with the wt. construct (data notshown).

Example 8

[0204] Replicons with Reporter Gene

[0205] This example illustrates an HCV replicon containing adaptivemutations and a reporter gene. A pHCVNeo17.wt derivative where the Neocoding region was substituted with that coding for human placentalsecretory alkaline phosphatase (pRBSEAP5.wt) and a derivative alsocontaining the N2041T and S2173F mutations (plasmid pRBSEAP5.m8) wereconstructed. RNAs transcribed from these plasmids were transfected in10IFN cells and their replication proficiency was assessed by measuringsecretion of alkaline phosphatase. Analysis of the kinetics of secretionsuggested that only plasmid pRBSEAP5.m8 was competent for replication(data not shown).

Example 9

[0206] SEQ. ID. Nos. 1 and 2

[0207] SEQ. ID. NOs. 1 and 2 are provided as follows: SEQ. ID. NO. 1MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPLPKARQPEGRAWAQPGYPWPLYGNEGLGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVLDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTIPASAYEVRNVSGVYHVTNDCSNASIVYEAADMIMHTPGCVPCVRENNSSRCWVALTPTLAARNASVPTTTIRRHVDLLVGAAALCSAMYVGDLCGSVFLVAQLFTFSPRRHETVQDCNCSIYPGHVTGHRMAWDMMMNWSPTAALVVSQLLRIPQAVVDMVAGAHWGVLAGLAYYSMVGNWAKVLIVMLLFAGVDGGTYVTGGTMAKNTLGITSLFSPGSSQKIQLVNTNGSWHINRTALNCNDSLNTGELAALFYVHKFNSSGCPERMASCSPIDAFAQGWGPITYNESHSSDQRPYCWHYAPRPCGIVPAAQVCGPVYCFTPSPVVVGTTDRFGVPTYSWGENETDVLLLNNTRPPQGNWFGCTWMNSTGFTKTCGGPPCNIGGIGNKTLTCPTDCFRKHPEATYTKCGSGPWLTPRCLVHYPYRLWHYPCTVNFTIFKVRMYVGGVEHRLEAACNWTRGERCNLEDRDRSELSPLLLSTTEWQVLPCSFITLPALSTGLIHLHQNVVDVQYLYGIGSAVVSFAIKWEYVLLLFLLLADARVCACLWMMLLIAQAEAALENLVVLNAASVAGAHGILSFLVFFCAAWYIKGRLVPGAAYALYGVWPLLLLLLALPPRAYAMDREMAASCGGAVFVGLILLTLSPHYKLFLARLLWWLQYFITRAEAHLQVWIPPLNVRGGRDAVILLTCAIHPELIFTITKILLAILGPLMVLQAGITKVPYFVRAHGLIRACMLVRKVAGGHYVQMALMKLAALTGTYVYDHLTPLRDWAHAGLRDLAVAVEPVVFSDMETKVITWGADTAACGDIILGLPVSARRGREIHLGPADSLEGQGWRLLAPLTAYSQQTRGLLGCIITSLTGRDRNQVEGEVQVVSTATQSFLATCVNGVCWTVYHGAGSKTLAGPKGPLTQMYTNVDQDLVGWQAPPGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPVSYLKGSSGGPLLCPSGHAVGIFRAAVCTRGVAKAVDFVPVESMETTMRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGAPITYSTYGKFLADGGCSGGAYDIIICDECHSTDSTTILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNLEEVALSSTGELPFYGKAIPIETLKGGRHLIFCHSKKKCDELAAKLSGLGLNAVAYYRGLDVSVIPTSGDVIVVATDALMTGFTGDFDSVLDCNTCVTQTVDFSLDPTFTIFITTVPQDAVSRSQRRGRTGRGRMGIYRFVTPGERPSGMFDSSVLCECYDAGCAWYELTPAETSVRLRAYLNTPGLPVCQDHLEFWESVFTGLTHIDAHFLSQTKQAGDNFPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTITTTHPITKYIMACMSADLEVVTSTWVLVGGVLAALAAYCLTTGSVVIVGRLILSGKPAIIPDREVLYREFDEMEECASHLPYIEQGMQLAEQFKQKAIGLLQTATKQAEAAAPVVESKWRTLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTASITSPLTTQHTLLFNILGGWVAAQLAPPSAASAFVGAGIAGAAVGSIGLGKVLVDILAGYGAGVAGALVAFKVMSGEMPSTEDLVNLLPAILSPGALVVGVVCAALLRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTQILSSLTITQLLKRLHQWINEDCSTPGSGSWLRDVWDWICTVLTDFKTWLQSKLLPRLPGVPFFSCQRGYKGVWRGDGIMQTTCPCGAQITGHVKNGSMRIVGPRTCSNTWHGTFPLNAYTTGPCTPSPAPNYSRALWRVAAEEYVEVTRVGDFHYVTGMTTDNVKCPCQVPAPEFFTEVDGVRLHRYAPACKPLLREEVTFLVGLNQYLVGSQLPCEPEPDVAVLTSMLTDPSHITAETAKRRLARGSPPSLASSSASQLSAPSLKATCTTRHDSPDADLIEANLLWRQEMGGNITRVESENKVVILDSFEPLQAEEDEREVSVPAEILRRSRKFPRAMPIWARPDYNPPLLESWKDPDYVPPVVHGCPLPPAKAPPIPPPRRKRTVVLSESTVSSALAELATKTFGSSESSAVDSGTATASPDQPSDDGDAGSDVESYSSMPPLEGEPGDPDLSDGSWSTVSEEASEDVVCCSMSYTWTGALITPCAAEETKLPINALSNSLLRHHNLVYAATTSRSASLRQKKVTFDRLQVLDDHYRDVLKEMKAKASTVKAKLLSVEEACKLTPPHSARSKFGYGAKDVRNLSSKAVNIIIRSVWKDLLEDTETPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSTLPQAVMGSSYGFQYSPGQRVEFLVNAWKAKKCPMGFAYDTRCFDSTVTENDIRVEESIYQCCDLAPEARQAIRSLTERLYIGGPLTNSKGQNCGYRRCRASGVLTTSCGNTLTCYLKAAAACRAAKLQDCTMLVCGDDLVVICESAGTQEDEASLRAFTEAMTRYSAPPGDPPKPEYDLELITSCSSNVSVAHDASGKRVYYLTRDPTTTPLARAAWETARHTPVNSWLGNIIMYAPTLWARMILMTHFFSILLAQEQLEKALDCQLYGACYSLEPLDLPQIIQRLHGLSAFSLHSYSPGEINRVASCLRKLGVPPLRVWRHRARSVRARLLSQGGRAATCGKYLFNWAVRTKLKLTPIPAASQLDLSSWFVAGYSGGDIYHSLSRARPRWFMWCL LLLSVGVGIYLLPNR SEQ.ID. NO. 2: gccagcccccgattgggggcgacactccaccatagatcactcccctgtgaggaactactgtcttcacgcagaaagcgtctagccatggcgttagtatgagtgtcgtgcagcctccaggaccccccctcccgggagagccatagtggtctgcggaaccggtgagtacaccggaattgccaggacgaccgggtcctttcttggatcaacccgctcaatgcctggagatttgggcgtgcccccgcgagactgctagccgagtagtgttgggtcgcgaaaggccttgtggtactgcctgatagggtgcttgcgagtgccccgggaggtctcgtagaccgtgcaccatgagcacgaatcctaaacctcaaagaaaaaccaaacgtaacaccaaccgccgcccacaggacgtcaagttcccgggcggtggtcagatcgtcggtggagtttacctgttgccgcgcaggggccccaggttgggtgtgcgcgcgactaggaagacttccgagcggtcgcaacctcgtggaaggcgacaacctatccccaaggctcgccagcccgagggtagggcctgggctcagcccgggtacccctggcccctctatggcaatgagggcttggggtgggcaggatggctcctgtcaccccgtggctctcggcctagttggggccccacggacccccggcgtaggtcgcgcaatttgggtaaggtcatcgataccctcacgtgcggcttcgccgatctcatggggtacattccgctcgtcggcgcccccctagggggcgctgccagggccctggcgcatggcgtccgggttctggaggacggcgtgaactatgcaacagggaatctgcccggttgctccttttctatcttccttttggctttgctgtcctgtttgaccatcccagcttccgcttatgaagtgcgcaacgtatccggagtgtaccatgtcacgaacgactgctccaacgcaagcattgtgtatgaggcagcggacatgatcatgcatacccccgggtgcgtgccctgcgttcgggagaacaactcctcccgctgctgggtagcgctcactcccacgctcgcggccaggaacgctagcgtccccactacgacgatacgacgccatgtcgatttgctcgttggggcggctgctctctgctccgctatgtacgtgggagatctctgcggatctgttttcctcgtcgcccagctgttcaccttctcgcctcgccggcacgagacagtacaggactgcaattgctcaatatatcccggccacgtgacaggtcaccgtatggcttgggatatgatgatgaactggtcacctacagcagccctagtggtatcgcagttactccggatcccacaagctgtcgtggatatggtggcgggggcccattggggagtcctagcgggccttgcctactattccatggtggggaactgggctaaggttctgattgtgatgctactctttgccggcgUgacgggggaacctatgtgacaggggggacgatggccaaaaacaccctcgggattacgtccctcttttcacccgggtcatcccagaaaatccagcttgtaaacaccaacggcagctggcacatcaacaggactgccctgaactgcaatgactccctcaacactgggttccttgctgcgctgttctacgtgcacaagttcaactcatctggatgcccagagcgcatggccagctgcagccccatcgacgcgttcgctcaggggtgggggcccatcacttacaatgagtcacacagctcggaccagaggccttattgttggcactacgcaccccggccgtgcggtatcgtacccgcggcgcaggtgtgtggtccagtgtactgcttcaccccaagccctgtcgtggtggggacgaccgaccggttcggcgtccctacgtacagttggggggagaatgagacggacgtgctgcttcttaacaacacgcggccgccgcaaggcaactggtttggctgtacatggatgaatagcactgggttcaccaagacgtgcgggggccccccgtgtaacatcggggggatcggcaataaaaccttgacctgccccacggactgcttccggaagcaccccgaggccacttacaccaagtgtggttcggggccttggttgacacccagatgcttggtccactacccatacaggctttggcactacccctgcactgtcaactttaccatcttcaaggttaggatgtacgtggggggagtggagcacaggctcgaagccgcatgcaattggactcgaggagagcgttgtaacctggaggacagggacagatcagagcttagcccgctgctgctgtctacaacggagtggcaggtattgccctgttccttcaccaccctaccggctctgtccactggtttgatccatctccatcagaacgtcgtggacgtacaatacctgtacggtatagggtcggcggttgtctcctttgcaatcaaatgggagtatgtcctgttgctcttccttcttctggcggacgcgcgcgtctgtgcctgcttgtggatgatgctgctgatagctcaagctgaggccgccctagagaacctggtggtcctcaacgcggcatccgtggccggggcgcatggcattctctccttcctcgtgttcttctgtgctgcctggtacatcaagggcaggctggtccctggggcggcatatgccctctacggcgtatggccgctactcctgctcctgctggcgttaccaccacgagcatacgccatggaccgggagatggcagcatcgtgcggaggcgcggttttcgtaggtctgatactcttgaccttgtcaccgcactataagctgttcctcgctaggctcatatggtggttacaatattttatcaccagggccgaggcacacttgcaagtgtggatcccccccctcaacgttcgggggggccgcgatgccgtcatcctcctcacgtgcgcgatccacccagagctaatctttaccatcaccaaaatcttgctcgccatactcggtccactcatggtgctccaggctggtataaccaaagtgccgtacttcgtgcgcgcacacgggctcattcgtgcatgcatgctggtgcggaaggttgctgggggtcattatgtccaaatggctctcatgaagttggccgcactgacaggtacgtacgtttatgaccatctcaccccactgcgggactgggcccacgcgggcctacgagaccttgcggtggcagttgagcccgtcgtcttctctgatatggagaccaaggttatcacctggggggcagacaccgcggcgtgtggggacatcatcttgggcctgcccgtctccgcccgcagggggagggagatacatctgggaccggcagacagccttgaagggcaggggtggcgactcctcgcgcctattacggcctactcccaacagacgcgaggcctacttggctgcatcatcactagcctcacaggccgggacaggaaccaggtcgagggggaggtccaagtggtctccaccgcaacacaatctttcctggcgacctgcgtcaatggcgtgtgttggactgtctatcatggtgccggctcaaagacccttgccggcccaaagggcccaatcacccaaatgtacaccaatgtggaccaggacctcgtcggctggcaagcgccccccggggcgcgttccttgacaccatgcacctgcggcagctcggacctttacttggtcacgaggcatgccgatgtcattccggtgcgccggcggggcgacagcagggggagcctactctcccccaggcccgtctcctacttgaagggctcttcgggcggtccactgctctgcccctcggggcacgctgtgggcatctttcgggctgccgtgtgcacccgaggggttgcgaaggcggtggactttgtacccgtcgagtctatggaaaccactatgcggtccccggtcttcacggacaactcgtcccctccggccgtaccgcagacattccaggtggcccatctacacgcccctactggtagcggcaagagcactaaggtgccggctgcgtatgcagcccaagggtataaggtgcttgtcctgaacccgtccgtcgccgccaccctaggtttcggggcgtatatgtctaaggcacatggtatcgaccctaacatcagaaccggggtaaggaccatcaccacgggtgcccccatcacgtactccacctatggcaagtttcttgccgacggtggttgctctgggggcgcctatgacatcataatatgtgatgagtgccactcaactgactcgaccactatcctgggcatcggcacagtcctggaccaagcggagacggctggagcgcgactcgtcgtgctcgccaccgctacgcctccgggatcggtcaccgtgccacatccaaacatcgaggaggtggctctgtccagcactggagaaatccccttttatggcaaagccatccccatcgagaccatcaagggggggaggcacctcattttctgccattccaagaagaaatgtgatgagctcgccgcgaagctgtccggcctcggactcaatgctgtagcatattaccggggccttgatgtatccgtcataccaactagcggagacgtcattgtcgagcaacggacgctctaatgacgggctttaccggcgatttcgactcagtgatcgactgcaatacatgtgtcacccagacagtcgacttcagcctggacccgaccttcaccattgagacgacgaccgtgccacaagacgcggtgtcacgctcgcagcggcgaggcaggactggtaggggcaggatgggcatttacaggtttgtgactccaggagaacggccctcgggcatgttcgattcctcggttctgtgcgagtgctatgacgcgggctgtgcttggtacgagctcacgcccgccgagacctcagttaggttgcgggcttacctaaacacaccagggttgcccgtctgccaggaccatctggagttctgggagagcgtctttacaggcctcacccacatagacgcccatttcttgtcccagactaagcaggcaggagacaacttcccctacctggtagcataccaggctacggtgtgcgccagggctcaggctccacctccatcgtgggaccaaatgtggaagtgtctcatacggctaaagcctacgctgcacgggccaacgcccctgctgtataggctgggagccgttcaaaacgaggttactaccacacaccccataaccaaatacatcatggcatgcatgtcggctgacctggaggtcgtcacgagcacctgggtgctggtaggcggagtcctagcagctctggccgcgtattgcctgacaacaggcagcgtggtcattgtgggcaggatcatcttgtccggaaagccggccatcattcccgacagggaagtcctttaccgggagttcgatgagatggaagagtgcgcctcacacctcccttacatcgaacagggaatgcagctcgccgaacaattcaaacagaaggcaatcgggttgctgcaaacagccaccaagcaagcggaggctgctgctcccgtggtggaatccaagtggcggaccctcgaagccttctgggcgaagcatatgtggaatttcatcagcgggatacaatatttagcaggcttgtccactctgcctggcaaccccgcgatagcatcactgatggcattcacagcctctatcaccagcccgctcaccacccaacatacccccctgtttaacatcctggggggatgggtggccgcccaacttgctcctcccagcgctgcttctgctttcgtaggcgccggcatcgctggagcggctgttggcagcataggccttgggaaggtgcttgtggatattttggcaggttatggagcaggggtggcaggcgcgctcgtggcctttaaggtcatgagcggcgagatgccctccaccgaggacctggttaacctactccctgctatcctctcccctggcgccctagtcgtcggggtcgtgtgcgcagcgatactgcgtcggcacgtgggcccaggggagggggctgtgcagtggatgaaccggctgatagcgttcgcttcgcggggtaaccacgtctcccccacgcactatgtgcctgagagcgacgctgcagcacgtgtcactcagatcctctctagtcttaccatcactcagctgctgaagaggcttcaccagtggatcaacgaggactgctccacgccatgctccggctcgtggctaagagatgtttgggattggatatgcacggtgttgactgatttcaagacctggctccagtccaagctcctgccgcgattgccgggagtccccttcttctcatgtcaacgtgggtacaagggagtctggcggggcgacggcatcatgcaaaccacctgcccatgtggagcacagatcaccggacatgtgaaaaacggttccatgaggatcgtggggcctaggacctgtagtaacacgtggcatggaacattccccattaacgcgtacaccacgggcccctgcacgccctccccggcgccaaattattctagggcgctgtggcgggtggctgctgaggagtacgtggaggttacgcgggtgggggatttccactacgtgacgggcatgaccactgacaacgtaaagtgcccgtgtcaggttccggcccccgaattcttcacagaagtggatggggtgcggttgcacaggtacgctccagcgtgcaaacccctcctacgggaggaggtcacattcctggtcgggctcaatcaatacctggttgggtcacagctcccatgcgagcccgaaccggacgtagcagtgctcacttccatgctcaccgacccctcccacattacggcggagacggctaagcgtaggctggccaggggatctcccccctccttggccagctcatcagctagccagctgtctgcgccttccttgaaggcaacatgcactacccgtcatgactccccggacgctgacctcatcgaggccaacctcctgtggcggcaggagatgggcgggaacatcacccgcgtggagtcagaaaataaggtagtaattttggactctttcgagccgctccaagcggaggaggatgagagggaagtatccgttccggcggagatcctgcggaggtccaggaaattccctcgagcgatgcccatatgggcacgcccggattacaaccctccactgttagagtcctggaaggacccggactacgtccctccagtggtacacgggtgtccattgccgcctgccaaggcccctccgataccacctccacggaggaagaggacggttgtcctgtcagaatctaccgtgtcttctgccttggcggagctcgccacaaagaccttcggcagctccgaatcgtcggccgtcgacagcggcacggcaacggcctctcctgaccagccctccgacgacggcgacgcgggatccgacgttgagtcgtactcctccatgcccccccttgagggggagccgggggatcccgatctcagcgacgggtcttggtctaccgtaagcgaggaggctagtgaggacgtcgtctgctgctcgatgtcctacacatggacaggcgccctgatcacgccatgcgctgcggaggaaaccaagctgcccatcaatgcactgagcaactctttgctccgtcaccacaacttggtctatgctacaacatctcgcagcgcaagcctgcggcagaagaaggtcacctttgacagactgcaggtcctggacgaccactaccgggacgtgctcaaggagatgaaggcgaaggcgtccacagttaaggctaaacttctatccgtggaggaagcctgtaagctgacgcccccacattcggccagatctaaatttggctatggggcaaaggacgtccggaacctatccagcaaggccgttaaccacatccgctccgtgtggaaggacttgctggaagacactgagacaccaattgacaccaccatcatggcaaaaaatgaggttttctgcgtccaaccagagaaggggggccgcaagccagctcgccttatcgtattcccagatttgggggttcgtgtgtgcgagaaaatggccctttacgatgtggtctccaccctccctcaggccgtgatgggctcttcatacggattccaatactctcctggacagcgggtcgagttcctggtgaatgcctggaaagcgaagaaatgccctatgggcttcgcatatgacacccgctgttttgactcaacggtcactgagaatgacatccgtgttgaggagtcaatctaccaatgttgtgacttggcccccgaagccagacaggccataaggtcgctcacagagcggctttacatcgggggccccctgactaattctaaagggcagaactgcggctatcgccggtgccgcgcgagcggtgtactgacgaccagctgcggtaataccctcacatgttacttgaaggccgctgcggcctgtcgagctgcgaagctccaggactgcacgatgctcgtatgcggagacgaccttgtcgttatctgtgaaagcgcggggacccaagaggacgaggcgagcctacgggccttcacggaggctatgactagatactctgccccccctggggacccgcccaaaccagaatacgacttggagttgataacatcatgctcctccaatgtgtcagtcgcgcacgatgcatctggcaaaagggtgtactatctcacccgtgaccccaccaccccccttgcgcgggctgcgtgggagacagctagacacactccagtcaattcctggctaggcaacatcatcatgtatgcgcccaccttgtgggcaaggatgatcctgatgactcatttcttctccatccttctagctcaggaacaacttgaaaaagccctagattgtcagatctacggggcctgttactccattgagccacttgacctacctcagatcattcaacgactccatggccttagcgcattttcactccatagttactctccaggtgagatcaatagggtggcttcatgcctcaggaaacttggggtaccgcccttgcgagtctggagacatcgggccagaagtgtccgcgctaggctactgtcccagggggggagggctgccacttgtggcaagtacctcttcaactgggcagtaaggaccaagctcaaactcactccaatcccggctgcgtcccagttggatttatccagctggttcgttgctggttacagcgggggagacatatatcacagcctgtctcgtgcccgaccccgctggttcatgtggtgcctactcctactttctgtaggggtaggcatctatctactccccaaccgatgaacggggagctaaacactccaggccaataggccatcctgtttttttccctttttttttttcttttttttttttttttttttttttttttttttttctcctttttttttcctctttttttccttttctttcctttggtggctccatcttagccctagtcacggctagctgtgaaaggtccgtgagccgcttgactgcagagagtgctgatactggcctctctgcagatca agt

[0208] Other embodiments are within the following claims. While severalembodiments have been shown and described, various modifications may bemade without departing from the spirit and scope of the presentinvention.

1 13 1 3010 PRT Con 1 HCV isolate nucleic acid 1 Met Ser Thr Asn Pro LysPro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg Pro Gln AspVal Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val Tyr Leu LeuPro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg Lys Thr SerGlu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 Ile Pro Lys Ala ArgGln Pro Glu Gly Arg Ala Trp Ala Gln Pro Gly 65 70 75 80 Tyr Pro Trp ProLeu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp 85 90 95 Leu Leu Ser ProArg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Arg Arg ArgSer Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 Gly PheAla Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 GlyGly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 145 150 155160 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165170 175 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr180 185 190 Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr Asn Asp CysSer 195 200 205 Asn Ala Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met HisThr Pro 210 215 220 Gly Cys Val Pro Cys Val Arg Glu Asn Asn Ser Ser ArgCys Trp Val 225 230 235 240 Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn AlaSer Val Pro Thr Thr 245 250 255 Thr Ile Arg Arg His Val Asp Leu Leu ValGly Ala Ala Ala Leu Cys 260 265 270 Ser Ala Met Tyr Val Gly Asp Leu CysGly Ser Val Phe Leu Val Ala 275 280 285 Gln Leu Phe Thr Phe Ser Pro ArgArg His Glu Thr Val Gln Asp Cys 290 295 300 Asn Cys Ser Ile Tyr Pro GlyHis Val Thr Gly His Arg Met Ala Trp 305 310 315 320 Asp Met Met Met AsnTrp Ser Pro Thr Ala Ala Leu Val Val Ser Gln 325 330 335 Leu Leu Arg IlePro Gln Ala Val Val Asp Met Val Ala Gly Ala His 340 345 350 Trp Gly ValLeu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp 355 360 365 Ala LysVal Leu Ile Val Met Leu Leu Phe Ala Gly Val Asp Gly Gly 370 375 380 ThrTyr Val Thr Gly Gly Thr Met Ala Lys Asn Thr Leu Gly Ile Thr 385 390 395400 Ser Leu Phe Ser Pro Gly Ser Ser Gln Lys Ile Gln Leu Val Asn Thr 405410 415 Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser420 425 430 Leu Asn Thr Gly Phe Leu Ala Ala Leu Phe Tyr Val His Lys PheAsn 435 440 445 Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Ser Pro IleAsp Ala 450 455 460 Phe Ala Gln Gly Trp Gly Pro Ile Thr Tyr Asn Glu SerHis Ser Ser 465 470 475 480 Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala ProArg Pro Cys Gly Ile 485 490 495 Val Pro Ala Ala Gln Val Cys Gly Pro ValTyr Cys Phe Thr Pro Ser 500 505 510 Pro Val Val Val Gly Thr Thr Asp ArgPhe Gly Val Pro Thr Tyr Ser 515 520 525 Trp Gly Glu Asn Glu Thr Asp ValLeu Leu Leu Asn Asn Thr Arg Pro 530 535 540 Pro Gln Gly Asn Trp Phe GlyCys Thr Trp Met Asn Ser Thr Gly Phe 545 550 555 560 Thr Lys Thr Cys GlyGly Pro Pro Cys Asn Ile Gly Gly Ile Gly Asn 565 570 575 Lys Thr Leu ThrCys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala 580 585 590 Thr Tyr ThrLys Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu 595 600 605 Val HisTyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe 610 615 620 ThrIle Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Leu 625 630 635640 Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asn Leu Glu Asp 645650 655 Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp660 665 670 Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser ThrGly 675 680 685 Leu Ile His Leu His Gln Asn Val Val Asp Val Gln Tyr LeuTyr Gly 690 695 700 Ile Gly Ser Ala Val Val Ser Phe Ala Ile Lys Trp GluTyr Val Leu 705 710 715 720 Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg ValCys Ala Cys Leu Trp 725 730 735 Met Met Leu Leu Ile Ala Gln Ala Glu AlaAla Leu Glu Asn Leu Val 740 745 750 Val Leu Asn Ala Ala Ser Val Ala GlyAla His Gly Ile Leu Ser Phe 755 760 765 Leu Val Phe Phe Cys Ala Ala TrpTyr Ile Lys Gly Arg Leu Val Pro 770 775 780 Gly Ala Ala Tyr Ala Leu TyrGly Val Trp Pro Leu Leu Leu Leu Leu 785 790 795 800 Leu Ala Leu Pro ProArg Ala Tyr Ala Met Asp Arg Glu Met Ala Ala 805 810 815 Ser Cys Gly GlyAla Val Phe Val Gly Leu Ile Leu Leu Thr Leu Ser 820 825 830 Pro His TyrLys Leu Phe Leu Ala Arg Leu Ile Trp Trp Leu Gln Tyr 835 840 845 Phe IleThr Arg Ala Glu Ala His Leu Gln Val Trp Ile Pro Pro Leu 850 855 860 AsnVal Arg Gly Gly Arg Asp Ala Val Ile Leu Leu Thr Cys Ala Ile 865 870 875880 His Pro Glu Leu Ile Phe Thr Ile Thr Lys Ile Leu Leu Ala Ile Leu 885890 895 Gly Pro Leu Met Val Leu Gln Ala Gly Ile Thr Lys Val Pro Tyr Phe900 905 910 Val Arg Ala His Gly Leu Ile Arg Ala Cys Met Leu Val Arg LysVal 915 920 925 Ala Gly Gly His Tyr Val Gln Met Ala Leu Met Lys Leu AlaAla Leu 930 935 940 Thr Gly Thr Tyr Val Tyr Asp His Leu Thr Pro Leu ArgAsp Trp Ala 945 950 955 960 His Ala Gly Leu Arg Asp Leu Ala Val Ala ValGlu Pro Val Val Phe 965 970 975 Ser Asp Met Glu Thr Lys Val Ile Thr TrpGly Ala Asp Thr Ala Ala 980 985 990 Cys Gly Asp Ile Ile Leu Gly Leu ProVal Ser Ala Arg Arg Gly Arg 995 1000 1005 Glu Ile His Leu Gly Pro AlaAsp Ser Leu Glu Gly Gln Gly Trp Arg 1010 1015 1020 Leu Leu Ala Pro IleThr Ala Tyr Ser Gln Gln Thr Arg Gly Leu Leu 1025 1030 1035 1040 Gly CysIle Ile Thr Ser Leu Thr Gly Arg Asp Arg Asn Gln Val Glu 1045 1050 1055Gly Glu Val Gln Val Val Ser Thr Ala Thr Gln Ser Phe Leu Ala Thr 10601065 1070 Cys Val Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly SerLys 1075 1080 1085 Thr Leu Ala Gly Pro Lys Gly Pro Ile Thr Gln Met TyrThr Asn Val 1090 1095 1100 Asp Gln Asp Leu Val Gly Trp Gln Ala Pro ProGly Ala Arg Ser Leu 1105 1110 1115 1120 Thr Pro Cys Thr Cys Gly Ser SerAsp Leu Tyr Leu Val Thr Arg His 1125 1130 1135 Ala Asp Val Ile Pro ValArg Arg Arg Gly Asp Ser Arg Gly Ser Leu 1140 1145 1150 Leu Ser Pro ArgPro Val Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro 1155 1160 1165 Leu LeuCys Pro Ser Gly His Ala Val Gly Ile Phe Arg Ala Ala Val 1170 1175 1180Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Val Pro Val Glu Ser 11851190 1195 1200 Met Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn SerSer Pro 1205 1210 1215 Pro Ala Val Pro Gln Thr Phe Gln Val Ala His LeuHis Ala Pro Thr 1220 1225 1230 Gly Ser Gly Lys Ser Thr Lys Val Pro AlaAla Tyr Ala Ala Gln Gly 1235 1240 1245 Tyr Lys Val Leu Val Leu Asn ProSer Val Ala Ala Thr Leu Gly Phe 1250 1255 1260 Gly Ala Tyr Met Ser LysAla His Gly Ile Asp Pro Asn Ile Arg Thr 1265 1270 1275 1280 Gly Val ArgThr Ile Thr Thr Gly Ala Pro Ile Thr Tyr Ser Thr Tyr 1285 1290 1295 GlyLys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile 1300 13051310 Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ser Thr Thr Ile Leu Gly1315 1320 1325 Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala ArgLeu Val 1330 1335 1340 Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Val ThrVal Pro His Pro 1345 1350 1355 1360 Asn Ile Glu Glu Val Ala Leu Ser SerThr Gly Glu Ile Pro Phe Tyr 1365 1370 1375 Gly Lys Ala Ile Pro Ile GluThr Ile Lys Gly Gly Arg His Leu Ile 1380 1385 1390 Phe Cys His Ser LysLys Lys Cys Asp Glu Leu Ala Ala Lys Leu Ser 1395 1400 1405 Gly Leu GlyLeu Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser 1410 1415 1420 ValIle Pro Thr Ser Gly Asp Val Ile Val Val Ala Thr Asp Ala Leu 1425 14301435 1440 Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val Ile Asp Cys AsnThr 1445 1450 1455 Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp Pro ThrPhe Thr Ile 1460 1465 1470 Glu Thr Thr Thr Val Pro Gln Asp Ala Val SerArg Ser Gln Arg Arg 1475 1480 1485 Gly Arg Thr Gly Arg Gly Arg Met GlyIle Tyr Arg Phe Val Thr Pro 1490 1495 1500 Gly Glu Arg Pro Ser Gly MetPhe Asp Ser Ser Val Leu Cys Glu Cys 1505 1510 1515 1520 Tyr Asp Ala GlyCys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Ser 1525 1530 1535 Val ArgLeu Arg Ala Tyr Leu Asn Thr Pro Gly Leu Pro Val Cys Gln 1540 1545 1550Asp His Leu Glu Phe Trp Glu Ser Val Phe Thr Gly Leu Thr His Ile 15551560 1565 Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ala Gly Asp Asn PhePro 1570 1575 1580 Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg AlaGln Ala Pro 1585 1590 1595 1600 Pro Pro Ser Trp Asp Gln Met Trp Lys CysLeu Ile Arg Leu Lys Pro 1605 1610 1615 Thr Leu His Gly Pro Thr Pro LeuLeu Tyr Arg Leu Gly Ala Val Gln 1620 1625 1630 Asn Glu Val Thr Thr ThrHis Pro Ile Thr Lys Tyr Ile Met Ala Cys 1635 1640 1645 Met Ser Ala AspLeu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly 1650 1655 1660 Gly ValLeu Ala Ala Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val 1665 1670 16751680 Val Ile Val Gly Arg Ile Ile Leu Ser Gly Lys Pro Ala Ile Ile Pro1685 1690 1695 Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu GluCys Ala 1700 1705 1710 Ser His Leu Pro Tyr Ile Glu Gln Gly Met Gln LeuAla Glu Gln Phe 1715 1720 1725 Lys Gln Lys Ala Ile Gly Leu Leu Gln ThrAla Thr Lys Gln Ala Glu 1730 1735 1740 Ala Ala Ala Pro Val Val Glu SerLys Trp Arg Thr Leu Glu Ala Phe 1745 1750 1755 1760 Trp Ala Lys His MetTrp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala 1765 1770 1775 Gly Leu SerThr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala 1780 1785 1790 PheThr Ala Ser Ile Thr Ser Pro Leu Thr Thr Gln His Thr Leu Leu 1795 18001805 Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala Pro Pro Ser1810 1815 1820 Ala Ala Ser Ala Phe Val Gly Ala Gly Ile Ala Gly Ala AlaVal Gly 1825 1830 1835 1840 Ser Ile Gly Leu Gly Lys Val Leu Val Asp IleLeu Ala Gly Tyr Gly 1845 1850 1855 Ala Gly Val Ala Gly Ala Leu Val AlaPhe Lys Val Met Ser Gly Glu 1860 1865 1870 Met Pro Ser Thr Glu Asp LeuVal Asn Leu Leu Pro Ala Ile Leu Ser 1875 1880 1885 Pro Gly Ala Leu ValVal Gly Val Val Cys Ala Ala Ile Leu Arg Arg 1890 1895 1900 His Val GlyPro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile 1905 1910 1915 1920Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro 19251930 1935 Glu Ser Asp Ala Ala Ala Arg Val Thr Gln Ile Leu Ser Ser LeuThr 1940 1945 1950 Ile Thr Gln Leu Leu Lys Arg Leu His Gln Trp Ile AsnGlu Asp Cys 1955 1960 1965 Ser Thr Pro Cys Ser Gly Ser Trp Leu Arg AspVal Trp Asp Trp Ile 1970 1975 1980 Cys Thr Val Leu Thr Asp Phe Lys ThrTrp Leu Gln Ser Lys Leu Leu 1985 1990 1995 2000 Pro Arg Leu Pro Gly ValPro Phe Phe Ser Cys Gln Arg Gly Tyr Lys 2005 2010 2015 Gly Val Trp ArgGly Asp Gly Ile Met Gln Thr Thr Cys Pro Cys Gly 2020 2025 2030 Ala GlnIle Thr Gly His Val Lys Asn Gly Ser Met Arg Ile Val Gly 2035 2040 2045Pro Arg Thr Cys Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn Ala 20502055 2060 Tyr Thr Thr Gly Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr SerArg 2065 2070 2075 2080 Ala Leu Trp Arg Val Ala Ala Glu Glu Tyr Val GluVal Thr Arg Val 2085 2090 2095 Gly Asp Phe His Tyr Val Thr Gly Met ThrThr Asp Asn Val Lys Cys 2100 2105 2110 Pro Cys Gln Val Pro Ala Pro GluPhe Phe Thr Glu Val Asp Gly Val 2115 2120 2125 Arg Leu His Arg Tyr AlaPro Ala Cys Lys Pro Leu Leu Arg Glu Glu 2130 2135 2140 Val Thr Phe LeuVal Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu 2145 2150 2155 2160 ProCys Glu Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr 2165 21702175 Asp Pro Ser His Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg2180 2185 2190 Gly Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln LeuSer Ala 2195 2200 2205 Pro Ser Leu Lys Ala Thr Cys Thr Thr Arg His AspSer Pro Asp Ala 2210 2215 2220 Asp Leu Ile Glu Ala Asn Leu Leu Trp ArgGln Glu Met Gly Gly Asn 2225 2230 2235 2240 Ile Thr Arg Val Glu Ser GluAsn Lys Val Val Ile Leu Asp Ser Phe 2245 2250 2255 Glu Pro Leu Gln AlaGlu Glu Asp Glu Arg Glu Val Ser Val Pro Ala 2260 2265 2270 Glu Ile LeuArg Arg Ser Arg Lys Phe Pro Arg Ala Met Pro Ile Trp 2275 2280 2285 AlaArg Pro Asp Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asp Pro 2290 22952300 Asp Tyr Val Pro Pro Val Val His Gly Cys Pro Leu Pro Pro Ala Lys2305 2310 2315 2320 Ala Pro Pro Ile Pro Pro Pro Arg Arg Lys Arg Thr ValVal Leu Ser 2325 2330 2335 Glu Ser Thr Val Ser Ser Ala Leu Ala Glu LeuAla Thr Lys Thr Phe 2340 2345 2350 Gly Ser Ser Glu Ser Ser Ala Val AspSer Gly Thr Ala Thr Ala Ser 2355 2360 2365 Pro Asp Gln Pro Ser Asp AspGly Asp Ala Gly Ser Asp Val Glu Ser 2370 2375 2380 Tyr Ser Ser Met ProPro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu 2385 2390 2395 2400 Ser AspGly Ser Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val 2405 2410 2415Val Cys Cys Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro 24202425 2430 Cys Ala Ala Glu Glu Thr Lys Leu Pro Ile Asn Ala Leu Ser AsnSer 2435 2440 2445 Leu Leu Arg His His Asn Leu Val Tyr Ala Thr Thr SerArg Ser Ala 2450 2455 2460 Ser Leu Arg Gln Lys Lys Val Thr Phe Asp ArgLeu Gln Val Leu Asp 2465 2470 2475 2480 Asp His Tyr Arg Asp Val Leu LysGlu Met Lys Ala Lys Ala Ser Thr 2485 2490 2495 Val Lys Ala Lys Leu LeuSer Val Glu Glu Ala Cys Lys Leu Thr Pro 2500 2505 2510 Pro His Ser AlaArg Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg 2515 2520 2525 Asn LeuSer Ser Lys Ala Val Asn His Ile Arg Ser Val Trp Lys Asp 2530 2535 2540Leu Leu Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala Lys 25452550 2555 2560 Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly Gly Arg LysPro Ala 2565 2570 2575 Arg Leu Ile Val Phe Pro Asp Leu Gly Val Arg ValCys Glu Lys Met 2580 2585 2590 Ala Leu Tyr Asp Val Val Ser Thr Leu ProGln Ala Val Met Gly Ser 2595 2600 2605 Ser Tyr Gly Phe Gln Tyr Ser ProGly Gln Arg Val Glu Phe Leu Val 2610 2615 2620 Asn Ala Trp Lys Ala LysLys Cys Pro Met Gly Phe Ala Tyr Asp Thr 2625 2630 2635 2640 Arg Cys PheAsp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu 2645 2650 2655 SerIle Tyr Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile 2660 26652670 Arg Ser Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser2675 2680 2685 Lys Gly Gln Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser GlyVal Leu 2690 2695 2700 Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr LeuLys Ala Ala Ala 2705 2710 2715 2720 Ala Cys Arg Ala Ala Lys Leu Gln AspCys Thr Met Leu Val Cys Gly 2725 2730 2735 Asp Asp Leu Val Val Ile CysGlu Ser Ala Gly Thr Gln Glu Asp Glu 2740 2745 2750 Ala Ser Leu Arg AlaPhe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro 2755 2760 2765 Pro Gly AspPro Pro Lys Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser 2770 2775 2780 CysSer Ser Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val 2785 27902795 2800 Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala AlaTrp 2805 2810 2815 Glu Thr Ala Arg His Thr Pro Val Asn Ser Trp Leu GlyAsn Ile Ile 2820 2825 2830 Met Tyr Ala Pro Thr Leu Trp Ala Arg Met IleLeu Met Thr His Phe 2835 2840 2845 Phe Ser Ile Leu Leu Ala Gln Glu GlnLeu Glu Lys Ala Leu Asp Cys 2850 2855 2860 Gln Ile Tyr Gly Ala Cys TyrSer Ile Glu Pro Leu Asp Leu Pro Gln 2865 2870 2875 2880 Ile Ile Gln ArgLeu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr 2885 2890 2895 Ser ProGly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly 2900 2905 2910Val Pro Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala 29152920 2925 Arg Leu Leu Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys TyrLeu 2930 2935 2940 Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr ProIle Pro Ala 2945 2950 2955 2960 Ala Ser Gln Leu Asp Leu Ser Ser Trp PheVal Ala Gly Tyr Ser Gly 2965 2970 2975 Gly Asp Ile Tyr His Ser Leu SerArg Ala Arg Pro Arg Trp Phe Met 2980 2985 2990 Trp Cys Leu Leu Leu LeuSer Val Gly Val Gly Ile Tyr Leu Leu Pro 2995 3000 3005 Asn Arg 3010 29605 DNA Con 1 HCV isolate amino acid 2 gccagccccc gattgggggc gacactccaccatagatcac tcccctgtga ggaactactg 60 tcttcacgca gaaagcgtct agccatggcgttagtatgag tgtcgtgcag cctccaggac 120 cccccctccc gggagagcca tagtggtctgcggaaccggt gagtacaccg gaattgccag 180 gacgaccggg tcctttcttg gatcaacccgctcaatgcct ggagatttgg gcgtgccccc 240 gcgagactgc tagccgagta gtgttgggtcgcgaaaggcc ttgtggtact gcctgatagg 300 gtgcttgcga gtgccccggg aggtctcgtagaccgtgcac catgagcacg aatcctaaac 360 ctcaaagaaa aaccaaacgt aacaccaaccgccgcccaca ggacgtcaag ttcccgggcg 420 gtggtcagat cgtcggtgga gtttacctgttgccgcgcag gggccccagg ttgggtgtgc 480 gcgcgactag gaagacttcc gagcggtcgcaacctcgtgg aaggcgacaa cctatcccca 540 aggctcgcca gcccgagggt agggcctgggctcagcccgg gtacccctgg cccctctatg 600 gcaatgaggg cttggggtgg gcaggatggctcctgtcacc ccgtggctct cggcctagtt 660 ggggccccac ggacccccgg cgtaggtcgcgcaatttggg taaggtcatc gataccctca 720 cgtgcggctt cgccgatctc atggggtacattccgctcgt cggcgccccc ctagggggcg 780 ctgccagggc cctggcgcat ggcgtccgggttctggagga cggcgtgaac tatgcaacag 840 ggaatctgcc cggttgctcc ttttctatcttccttttggc tttgctgtcc tgtttgacca 900 tcccagcttc cgcttatgaa gtgcgcaacgtatccggagt gtaccatgtc acgaacgact 960 gctccaacgc aagcattgtg tatgaggcagcggacatgat catgcatacc cccgggtgcg 1020 tgccctgcgt tcgggagaac aactcctcccgctgctgggt agcgctcact cccacgctcg 1080 cggccaggaa cgctagcgtc cccactacgacgatacgacg ccatgtcgat ttgctcgttg 1140 gggcggctgc tctctgctcc gctatgtacgtgggagatct ctgcggatct gttttcctcg 1200 tcgcccagct gttcaccttc tcgcctcgccggcacgagac agtacaggac tgcaattgct 1260 caatatatcc cggccacgtg acaggtcaccgtatggcttg ggatatgatg atgaactggt 1320 cacctacagc agccctagtg gtatcgcagttactccggat cccacaagct gtcgtggata 1380 tggtggcggg ggcccattgg ggagtcctagcgggccttgc ctactattcc atggtgggga 1440 actgggctaa ggttctgatt gtgatgctactctttgccgg cgttgacggg ggaacctatg 1500 tgacaggggg gacgatggcc aaaaacaccctcgggattac gtccctcttt tcacccgggt 1560 catcccagaa aatccagctt gtaaacaccaacggcagctg gcacatcaac aggactgccc 1620 tgaactgcaa tgactccctc aacactgggttccttgctgc gctgttctac gtgcacaagt 1680 tcaactcatc tggatgccca gagcgcatggccagctgcag ccccatcgac gcgttcgctc 1740 aggggtgggg gcccatcact tacaatgagtcacacagctc ggaccagagg ccttattgtt 1800 ggcactacgc accccggccg tgcggtatcgtacccgcggc gcaggtgtgt ggtccagtgt 1860 actgcttcac cccaagccct gtcgtggtggggacgaccga ccggttcggc gtccctacgt 1920 acagttgggg ggagaatgag acggacgtgctgcttcttaa caacacgcgg ccgccgcaag 1980 gcaactggtt tggctgtaca tggatgaatagcactgggtt caccaagacg tgcgggggcc 2040 ccccgtgtaa catcgggggg atcggcaataaaaccttgac ctgccccacg gactgcttcc 2100 ggaagcaccc cgaggccact tacaccaagtgtggttcggg gccttggttg acacccagat 2160 gcttggtcca ctacccatac aggctttggcactacccctg cactgtcaac tttaccatct 2220 tcaaggttag gatgtacgtg gggggagtggagcacaggct cgaagccgca tgcaattgga 2280 ctcgaggaga gcgttgtaac ctggaggacagggacagatc agagcttagc ccgctgctgc 2340 tgtctacaac ggagtggcag gtattgccctgttccttcac caccctaccg gctctgtcca 2400 ctggtttgat ccatctccat cagaacgtcgtggacgtaca atacctgtac ggtatagggt 2460 cggcggttgt ctcctttgca atcaaatgggagtatgtcct gttgctcttc cttcttctgg 2520 cggacgcgcg cgtctgtgcc tgcttgtggatgatgctgct gatagctcaa gctgaggccg 2580 ccctagagaa cctggtggtc ctcaacgcggcatccgtggc cggggcgcat ggcattctct 2640 ccttcctcgt gttcttctgt gctgcctggtacatcaaggg caggctggtc cctggggcgg 2700 catatgccct ctacggcgta tggccgctactcctgctcct gctggcgtta ccaccacgag 2760 catacgccat ggaccgggag atggcagcatcgtgcggagg cgcggttttc gtaggtctga 2820 tactcttgac cttgtcaccg cactataagctgttcctcgc taggctcata tggtggttac 2880 aatattttat caccagggcc gaggcacacttgcaagtgtg gatccccccc ctcaacgttc 2940 gggggggccg cgatgccgtc atcctcctcacgtgcgcgat ccacccagag ctaatcttta 3000 ccatcaccaa aatcttgctc gccatactcggtccactcat ggtgctccag gctggtataa 3060 ccaaagtgcc gtacttcgtg cgcgcacacgggctcattcg tgcatgcatg ctggtgcgga 3120 aggttgctgg gggtcattat gtccaaatggctctcatgaa gttggccgca ctgacaggta 3180 cgtacgttta tgaccatctc accccactgcgggactgggc ccacgcgggc ctacgagacc 3240 ttgcggtggc agttgagccc gtcgtcttctctgatatgga gaccaaggtt atcacctggg 3300 gggcagacac cgcggcgtgt ggggacatcatcttgggcct gcccgtctcc gcccgcaggg 3360 ggagggagat acatctggga ccggcagacagccttgaagg gcaggggtgg cgactcctcg 3420 cgcctattac ggcctactcc caacagacgcgaggcctact tggctgcatc atcactagcc 3480 tcacaggccg ggacaggaac caggtcgagggggaggtcca agtggtctcc accgcaacac 3540 aatctttcct ggcgacctgc gtcaatggcgtgtgttggac tgtctatcat ggtgccggct 3600 caaagaccct tgccggccca aagggcccaatcacccaaat gtacaccaat gtggaccagg 3660 acctcgtcgg ctggcaagcg ccccccggggcgcgttcctt gacaccatgc acctgcggca 3720 gctcggacct ttacttggtc acgaggcatgccgatgtcat tccggtgcgc cggcggggcg 3780 acagcagggg gagcctactc tcccccaggcccgtctccta cttgaagggc tcttcgggcg 3840 gtccactgct ctgcccctcg gggcacgctgtgggcatctt tcgggctgcc gtgtgcaccc 3900 gaggggttgc gaaggcggtg gactttgtacccgtcgagtc tatggaaacc actatgcggt 3960 ccccggtctt cacggacaac tcgtcccctccggccgtacc gcagacattc caggtggccc 4020 atctacacgc ccctactggt agcggcaagagcactaaggt gccggctgcg tatgcagccc 4080 aagggtataa ggtgcttgtc ctgaacccgtccgtcgccgc caccctaggt ttcggggcgt 4140 atatgtctaa ggcacatggt atcgaccctaacatcagaac cggggtaagg accatcacca 4200 cgggtgcccc catcacgtac tccacctatggcaagtttct tgccgacggt ggttgctctg 4260 ggggcgccta tgacatcata atatgtgatgagtgccactc aactgactcg accactatcc 4320 tgggcatcgg cacagtcctg gaccaagcggagacggctgg agcgcgactc gtcgtgctcg 4380 ccaccgctac gcctccggga tcggtcaccgtgccacatcc aaacatcgag gaggtggctc 4440 tgtccagcac tggagaaatc cccttttatggcaaagccat ccccatcgag accatcaagg 4500 gggggaggca cctcattttc tgccattccaagaagaaatg tgatgagctc gccgcgaagc 4560 tgtccggcct cggactcaat gctgtagcatattaccgggg ccttgatgta tccgtcatac 4620 caactagcgg agacgtcatt gtcgtagcaacggacgctct aatgacgggc tttaccggcg 4680 atttcgactc agtgatcgac tgcaatacatgtgtcaccca gacagtcgac ttcagcctgg 4740 acccgacctt caccattgag acgacgaccgtgccacaaga cgcggtgtca cgctcgcagc 4800 ggcgaggcag gactggtagg ggcaggatgggcatttacag gtttgtgact ccaggagaac 4860 ggccctcggg catgttcgat tcctcggttctgtgcgagtg ctatgacgcg ggctgtgctt 4920 ggtacgagct cacgcccgcc gagacctcagttaggttgcg ggcttaccta aacacaccag 4980 ggttgcccgt ctgccaggac catctggagttctgggagag cgtctttaca ggcctcaccc 5040 acatagacgc ccatttcttg tcccagactaagcaggcagg agacaacttc ccctacctgg 5100 tagcatacca ggctacggtg tgcgccagggctcaggctcc acctccatcg tgggaccaaa 5160 tgtggaagtg tctcatacgg ctaaagcctacgctgcacgg gccaacgccc ctgctgtata 5220 ggctgggagc cgttcaaaac gaggttactaccacacaccc cataaccaaa tacatcatgg 5280 catgcatgtc ggctgacctg gaggtcgtcacgagcacctg ggtgctggta ggcggagtcc 5340 tagcagctct ggccgcgtat tgcctgacaacaggcagcgt ggtcattgtg ggcaggatca 5400 tcttgtccgg aaagccggcc atcattcccgacagggaagt cctttaccgg gagttcgatg 5460 agatggaaga gtgcgcctca cacctcccttacatcgaaca gggaatgcag ctcgccgaac 5520 aattcaaaca gaaggcaatc gggttgctgcaaacagccac caagcaagcg gaggctgctg 5580 ctcccgtggt ggaatccaag tggcggaccctcgaagcctt ctgggcgaag catatgtgga 5640 atttcatcag cgggatacaa tatttagcaggcttgtccac tctgcctggc aaccccgcga 5700 tagcatcact gatggcattc acagcctctatcaccagccc gctcaccacc caacataccc 5760 tcctgtttaa catcctgggg ggatgggtggccgcccaact tgctcctccc agcgctgctt 5820 ctgctttcgt aggcgccggc atcgctggagcggctgttgg cagcataggc cttgggaagg 5880 tgcttgtgga tattttggca ggttatggagcaggggtggc aggcgcgctc gtggccttta 5940 aggtcatgag cggcgagatg ccctccaccgaggacctggt taacctactc cctgctatcc 6000 tctcccctgg cgccctagtc gtcggggtcgtgtgcgcagc gatactgcgt cggcacgtgg 6060 gcccagggga gggggctgtg cagtggatgaaccggctgat agcgttcgct tcgcggggta 6120 accacgtctc ccccacgcac tatgtgcctgagagcgacgc tgcagcacgt gtcactcaga 6180 tcctctctag tcttaccatc actcagctgctgaagaggct tcaccagtgg atcaacgagg 6240 actgctccac gccatgctcc ggctcgtggctaagagatgt ttgggattgg atatgcacgg 6300 tgttgactga tttcaagacc tggctccagtccaagctcct gccgcgattg ccgggagtcc 6360 ccttcttctc atgtcaacgt gggtacaagggagtctggcg gggcgacggc atcatgcaaa 6420 ccacctgccc atgtggagca cagatcaccggacatgtgaa aaacggttcc atgaggatcg 6480 tggggcctag gacctgtagt aacacgtggcatggaacatt ccccattaac gcgtacacca 6540 cgggcccctg cacgccctcc ccggcgccaaattattctag ggcgctgtgg cgggtggctg 6600 ctgaggagta cgtggaggtt acgcgggtgggggatttcca ctacgtgacg ggcatgacca 6660 ctgacaacgt aaagtgcccg tgtcaggttccggcccccga attcttcaca gaagtggatg 6720 gggtgcggtt gcacaggtac gctccagcgtgcaaacccct cctacgggag gaggtcacat 6780 tcctggtcgg gctcaatcaa tacctggttgggtcacagct cccatgcgag cccgaaccgg 6840 acgtagcagt gctcacttcc atgctcaccgacccctccca cattacggcg gagacggcta 6900 agcgtaggct ggccagggga tctcccccctccttggccag ctcatcagct agccagctgt 6960 ctgcgccttc cttgaaggca acatgcactacccgtcatga ctccccggac gctgacctca 7020 tcgaggccaa cctcctgtgg cggcaggagatgggcgggaa catcacccgc gtggagtcag 7080 aaaataaggt agtaattttg gactctttcgagccgctcca agcggaggag gatgagaggg 7140 aagtatccgt tccggcggag atcctgcggaggtccaggaa attccctcga gcgatgccca 7200 tatgggcacg cccggattac aaccctccactgttagagtc ctggaaggac ccggactacg 7260 tccctccagt ggtacacggg tgtccattgccgcctgccaa ggcccctccg ataccacctc 7320 cacggaggaa gaggacggtt gtcctgtcagaatctaccgt gtcttctgcc ttggcggagc 7380 tcgccacaaa gaccttcggc agctccgaatcgtcggccgt cgacagcggc acggcaacgg 7440 cctctcctga ccagccctcc gacgacggcgacgcgggatc cgacgttgag tcgtactcct 7500 ccatgccccc ccttgagggg gagccgggggatcccgatct cagcgacggg tcttggtcta 7560 ccgtaagcga ggaggctagt gaggacgtcgtctgctgctc gatgtcctac acatggacag 7620 gcgccctgat cacgccatgc gctgcggaggaaaccaagct gcccatcaat gcactgagca 7680 actctttgct ccgtcaccac aacttggtctatgctacaac atctcgcagc gcaagcctgc 7740 ggcagaagaa ggtcaccttt gacagactgcaggtcctgga cgaccactac cgggacgtgc 7800 tcaaggagat gaaggcgaag gcgtccacagttaaggctaa acttctatcc gtggaggaag 7860 cctgtaagct gacgccccca cattcggccagatctaaatt tggctatggg gcaaaggacg 7920 tccggaacct atccagcaag gccgttaaccacatccgctc cgtgtggaag gacttgctgg 7980 aagacactga gacaccaatt gacaccaccatcatggcaaa aaatgaggtt ttctgcgtcc 8040 aaccagagaa ggggggccgc aagccagctcgccttatcgt attcccagat ttgggggttc 8100 gtgtgtgcga gaaaatggcc ctttacgatgtggtctccac cctccctcag gccgtgatgg 8160 gctcttcata cggattccaa tactctcctggacagcgggt cgagttcctg gtgaatgcct 8220 ggaaagcgaa gaaatgccct atgggcttcgcatatgacac ccgctgtttt gactcaacgg 8280 tcactgagaa tgacatccgt gttgaggagtcaatctacca atgttgtgac ttggcccccg 8340 aagccagaca ggccataagg tcgctcacagagcggcttta catcgggggc cccctgacta 8400 attctaaagg gcagaactgc ggctatcgccggtgccgcgc gagcggtgta ctgacgacca 8460 gctgcggtaa taccctcaca tgttacttgaaggccgctgc ggcctgtcga gctgcgaagc 8520 tccaggactg cacgatgctc gtatgcggagacgaccttgt cgttatctgt gaaagcgcgg 8580 ggacccaaga ggacgaggcg agcctacgggccttcacgga ggctatgact agatactctg 8640 ccccccctgg ggacccgccc aaaccagaatacgacttgga gttgataaca tcatgctcct 8700 ccaatgtgtc agtcgcgcac gatgcatctggcaaaagggt gtactatctc acccgtgacc 8760 ccaccacccc ccttgcgcgg gctgcgtgggagacagctag acacactcca gtcaattcct 8820 ggctaggcaa catcatcatg tatgcgcccaccttgtgggc aaggatgatc ctgatgactc 8880 atttcttctc catccttcta gctcaggaacaacttgaaaa agccctagat tgtcagatct 8940 acggggcctg ttactccatt gagccacttgacctacctca gatcattcaa cgactccatg 9000 gccttagcgc attttcactc catagttactctccaggtga gatcaatagg gtggcttcat 9060 gcctcaggaa acttggggta ccgcccttgcgagtctggag acatcgggcc agaagtgtcc 9120 gcgctaggct actgtcccag ggggggagggctgccacttg tggcaagtac ctcttcaact 9180 gggcagtaag gaccaagctc aaactcactccaatcccggc tgcgtcccag ttggatttat 9240 ccagctggtt cgttgctggt tacagcgggggagacatata tcacagcctg tctcgtgccc 9300 gaccccgctg gttcatgtgg tgcctactcctactttctgt aggggtaggc atctatctac 9360 tccccaaccg atgaacgggg agctaaacactccaggccaa taggccatcc tgtttttttc 9420 cctttttttt tttctttttt tttttttttttttttttttt ttttttttct cctttttttt 9480 tcctcttttt ttccttttct ttcctttggtggctccatct tagccctagt cacggctagc 9540 tgtgaaaggt ccgtgagccg cttgactgcagagagtgctg atactggcct ctctgcagat 9600 caagt 9605 3 10690 DNA pHCVNeo.17coding 3 gccagccccc gattgggggc gacactccac catagatcac tcccctgtgaggaactactg 60 tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcagcctccaggac 120 cccccctccc gggagagcca tagtggtctg cggaaccggt gagtacaccggaattgccag 180 gacgaccggg tcctttcttg gatcaacccg ctcaatgcct ggagatttgggcgtgccccc 240 gcgagactgc tagccgagta gtgttgggtc gcgaaaggcc ttgtggtactgcctgatagg 300 gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacgaatcctaaac 360 ctcaaagaaa aaccaaaggg cgcgccatga ttgaacaaga tggattgcacgcaggttctc 420 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagacaatcggctgct 480 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttctttttgtcaagaccg 540 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcgtggctggcca 600 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcgggaagggactggc 660 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgctcctgccgaga 720 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccggctacctgcc 780 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatggaagccggtc 840 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagccgaactgttcg 900 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccatggcgatgcct 960 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgactgtggccggc 1020 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatattgctgaagagc 1080 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgctcccgattcgc 1140 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agtttaaacagaccacaacg 1200 gtttccctct agcgggatca attccgcccc tctccctccc ccccccctaacgttactggc 1260 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttccaccatattg 1320 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgacgagcattcct 1380 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgtgaaggaagca 1440 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttgcaggcagcgg 1500 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtataagatacacct 1560 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtggaaagagtcaaa 1620 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggtaccccattgt 1680 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtcgaggttaaaa 1740 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaacacgataatacc 1800 atggcgccta ttacggccta ctcccaacag acgcgaggcc tacttggctgcatcatcact 1860 agcctcacag gccgggacag gaaccaggtc gagggggagg tccaagtggtctccaccgca 1920 acacaatctt tcctggcgac ctgcgtcaat ggcgtgtgtt ggactgtctatcatggtgcc 1980 ggctcaaaga cccttgccgg cccaaagggc ccaatcaccc aaatgtacaccaatgtggac 2040 caggacctcg tcggctggca agcgcccccc ggggcgcgtt ccttgacaccatgcacctgc 2100 ggcagctcgg acctttactt ggtcacgagg catgccgatg tcattccggtgcgccggcgg 2160 ggcgacagca gggggagcct actctccccc aggcccgtct cctacttgaagggctcttcg 2220 ggcggtccac tgctctgccc ctcggggcac gctgtgggca tctttcgggctgccgtgtgc 2280 acccgagggg ttgcgaaggc ggtggacttt gtacccgtcg agtctatggaaaccactatg 2340 cggtccccgg tcttcacgga caactcgtcc cctccggccg taccgcagacattccaggtg 2400 gcccatctac acgcccctac tggtagcggc aagagcacta aggtgccggctgcgtatgca 2460 gcccaagggt ataaggtgct tgtcctgaac ccgtccgtcg ccgccaccctaggtttcggg 2520 gcgtatatgt ctaaggcaca tggtatcgac cctaacatca gaaccggggtaaggaccatc 2580 accacgggtg cccccatcac gtactccacc tatggcaagt ttcttgccgacggtggttgc 2640 tctgggggcg cctatgacat cataatatgt gatgagtgcc actcaactgactcgaccact 2700 atcctgggca tcggcacagt cctggaccaa gcggagacgg ctggagcgcgactcgtcgtg 2760 ctcgccaccg ctacgcctcc gggatcggtc accgtgccac atccaaacatcgaggaggtg 2820 gctctgtcca gcactggaga aatccccttt tatggcaaag ccatccccatcgagaccatc 2880 aaggggggga ggcacctcat tttctgccat tccaagaaga aatgtgatgagctcgccgcg 2940 aagctgtccg gcctcggact caatgctgta gcatattacc ggggccttgatgtatccgtc 3000 ataccaacta gcggagacgt cattgtcgta gcaacggacg ctctaatgacgggctttacc 3060 ggcgatttcg actcagtgat cgactgcaat acatgtgtca cccagacagtcgacttcagc 3120 ctggacccga ccttcaccat tgagacgacg accgtgccac aagacgcggtgtcacgctcg 3180 cagcggcgag gcaggactgg taggggcagg atgggcattt acaggtttgtgactccagga 3240 gaacggccct cgggcatgtt cgattcctcg gttctgtgcg agtgctatgacgcgggctgt 3300 gcttggtacg agctcacgcc cgccgagacc tcagttaggt tgcgggcttacctaaacaca 3360 ccagggttgc ccgtctgcca ggaccatctg gagttctggg agagcgtctttacaggcctc 3420 acccacatag acgcccattt cttgtcccag actaagcagg caggagacaacttcccctac 3480 ctggtagcat accaggctac ggtgtgcgcc agggctcagg ctccacctccatcgtgggac 3540 caaatgtgga agtgtctcat acggctaaag cctacgctgc acgggccaacgcccctgctg 3600 tataggctgg gagccgttca aaacgaggtt actaccacac accccataaccaaatacatc 3660 atggcatgca tgtcggctga cctggaggtc gtcacgagca cctgggtgctggtaggcgga 3720 gtcctagcag ctctggccgc gtattgcctg acaacaggca gcgtggtcattgtgggcagg 3780 atcatcttgt ccggaaagcc ggccatcatt cccgacaggg aagtcctttaccgggagttc 3840 gatgagatgg aagagtgcgc ctcacacctc ccttacatcg aacagggaatgcagctcgcc 3900 gaacaattca aacagaaggc aatcgggttg ctgcaaacag ccaccaagcaagcggaggct 3960 gctgctcccg tggtggaatc caagtggcgg accctcgaag ccttctgggcgaagcatatg 4020 tggaatttca tcagcgggat acaatattta gcaggcttgt ccactctgcctggcaacccc 4080 gcgatagcat cactgatggc attcacagcc tctatcacca gcccgctcaccacccaacat 4140 accctcctgt ttaacatcct ggggggatgg gtggccgccc aacttgctcctcccagcgct 4200 gcttctgctt tcgtaggcgc cggcatcgct ggagcggctg ttggcagcataggccttggg 4260 aaggtgcttg tggatatttt ggcaggttat ggagcagggg tggcaggcgcgctcgtggcc 4320 tttaaggtca tgagcggcga gatgccctcc accgaggacc tggttaacctactccctgct 4380 atcctctccc ctggcgccct agtcgtcggg gtcgtgtgcg cagcgatactgcgtcggcac 4440 gtgggcccag gggagggggc tgtgcagtgg atgaaccggc tgatagcgttcgcttcgcgg 4500 ggtaaccacg tctcccccac gcactatgtg cctgagagcg acgctgcagcacgtgtcact 4560 cagatcctct ctagtcttac catcactcag ctgctgaaga ggcttcaccagtggatcaac 4620 gaggactgct ccacgccatg ctccggctcg tggctaagag atgtttgggattggatatgc 4680 acggtgttga ctgatttcaa gacctggctc cagtccaagc tcctgccgcgattgccggga 4740 gtccccttct tctcatgtca acgtgggtac aagggagtct ggcggggcgacggcatcatg 4800 caaaccacct gcccatgtgg agcacagatc accggacatg tgaaaaacggttccatgagg 4860 atcgtggggc ctaggacctg tagtaacacg tggcatggaa cattccccattaacgcgtac 4920 accacgggcc cctgcacgcc ctccccggcg ccaaattatt ctagggcgctgtggcgggtg 4980 gctgctgagg agtacgtgga ggttacgcgg gtgggggatt tccactacgtgacgggcatg 5040 accactgaca acgtaaagtg cccgtgtcag gttccggccc ccgaattcttcacagaagtg 5100 gatggggtgc ggttgcacag gtacgctcca gcgtgcaaac ccctcctacgggaggaggtc 5160 acattcctgg tcgggctcaa tcaatacctg gttgggtcac agctcccatgcgagcccgaa 5220 ccggacgtag cagtgctcac ttccatgctc accgacccct cccacattacggcggagacg 5280 gctaagcgta ggctggccag gggatctccc ccctccttgg ccagctcatcagctagccag 5340 ctgtctgcgc cttccttgaa ggcaacatgc actacccgtc atgactccccggacgctgac 5400 ctcatcgagg ccaacctcct gtggcggcag gagatgggcg ggaacatcacccgcgtggag 5460 tcagaaaata aggtagtaat tttggactct ttcgagccgc tccaagcggaggaggatgag 5520 agggaagtat ccgttccggc ggagatcctg cggaggtcca ggaaattccctcgagcgatg 5580 cccatatggg cacgcccgga ttacaaccct ccactgttag agtcctggaaggacccggac 5640 tacgtccctc cagtggtaca cgggtgtcca ttgccgcctg ccaaggcccctccgatacca 5700 cctccacgga ggaagaggac ggttgtcctg tcagaatcta ccgtgtcttctgccttggcg 5760 gagctcgcca caaagacctt cggcagctcc gaatcgtcgg ccgtcgacagcggcacggca 5820 acggcctctc ctgaccagcc ctccgacgac ggcgacgcgg gatccgacgttgagtcgtac 5880 tcctccatgc ccccccttga gggggagccg ggggatcccg atctcagcgacgggtcttgg 5940 tctaccgtaa gcgaggaggc tagtgaggac gtcgtctgct gctcgatgtcctacacatgg 6000 acaggcgccc tgatcacgcc atgcgctgcg gaggaaacca agctgcccatcaatgcactg 6060 agcaactctt tgctccgtca ccacaacttg gtctatgcta caacatctcgcagcgcaagc 6120 ctgcggcaga agaaggtcac ctttgacaga ctgcaggtcc tggacgaccactaccgggac 6180 gtgctcaagg agatgaaggc gaaggcgtcc acagttaagg ctaaacttctatccgtggag 6240 gaagcctgta agctgacgcc cccacattcg gccagatcta aatttggctatggggcaaag 6300 gacgtccgga acctatccag caaggccgtt aaccacatcc gctccgtgtggaaggacttg 6360 ctggaagaca ctgagacacc aattgacacc accatcatgg caaaaaatgaggttttctgc 6420 gtccaaccag agaagggggg ccgcaagcca gctcgcctta tcgtattcccagatttgggg 6480 gttcgtgtgt gcgagaaaat ggccctttac gatgtggtct ccaccctccctcaggccgtg 6540 atgggctctt catacggatt ccaatactct cctggacagc gggtcgagttcctggtgaat 6600 gcctggaaag cgaagaaatg ccctatgggc ttcgcatatg acacccgctgttttgactca 6660 acggtcactg agaatgacat ccgtgttgag gagtcaatct accaatgttgtgacttggcc 6720 cccgaagcca gacaggccat aaggtcgctc acagagcggc tttacatcgggggccccctg 6780 actaattcta aagggcagaa ctgcggctat cgccggtgcc gcgcgagcggtgtactgacg 6840 accagctgcg gtaataccct cacatgttac ttgaaggccg ctgcggcctgtcgagctgcg 6900 aagctccagg actgcacgat gctcgtatgc ggagacgacc ttgtcgttatctgtgaaagc 6960 gcggggaccc aagaggacga ggcgagccta cgggccttca cggaggctatgactagatac 7020 tctgcccccc ctggggaccc gcccaaacca gaatacgact tggagttgataacatcatgc 7080 tcctccaatg tgtcagtcgc gcacgatgca tctggcaaaa gggtgtactatctcacccgt 7140 gaccccacca ccccccttgc gcgggctgcg tgggagacag ctagacacactccagtcaat 7200 tcctggctag gcaacatcat catgtatgcg cccaccttgt gggcaaggatgatcctgatg 7260 actcatttct tctccatcct tctagctcag gaacaacttg aaaaagccctagattgtcag 7320 atctacgggg cctgttactc cattgagcca cttgacctac ctcagatcattcaacgactc 7380 catggcctta gcgcattttc actccatagt tactctccag gtgagatcaatagggtggct 7440 tcatgcctca ggaaacttgg ggtaccgccc ttgcgagtct ggagacatcgggccagaagt 7500 gtccgcgcta ggctactgtc ccaggggggg agggctgcca cttgtggcaagtacctcttc 7560 aactgggcag taaggaccaa gctcaaactc actccaatcc cggctgcgtcccagttggat 7620 ttatccagct ggttcgttgc tggttacagc gggggagaca tatatcacagcctgtctcgt 7680 gcccgacccc gctggttcat gtggtgccta ctcctacttt ctgtaggggtaggcatctat 7740 ctactcccca accgatgaac ggggagctaa acactccagg ccaataggccatcctgtttt 7800 tttccctttt tttttttctt tttttttttt tttttttttt ttttttttttttctcctttt 7860 tttttcctct ttttttcctt ttctttcctt tggtggctcc atcttagccctagtcacggc 7920 tagctgtgaa aggtccgtga gccgcttgac tgcagagagt gctgatactggcctctctgc 7980 agatcaagta cttctagaga attctagctt ggcgtaatca tggtcatagctgtttcctgt 8040 gtgaaattgt tatcagctca caattccaca caacatacga gccggaagcataaagtgtaa 8100 agcctgggat gcctaatgag tgagctaact cacattagtt gcgttgcgctcactgcccgc 8160 tttccagtcg ggaaacctgt cgtgccagct ccattagtga atcgtccaacgcacggggag 8220 aggcggtttg cgtattgggc gcacttccgc ttcctcgctc actgactcgctgcgctcgtt 8280 cgttcggctg cggcgagccg tatcagctca ctcaaaggcg gtaatacggttatccacaga 8340 atcaggggat aacgcaggaa agaccatgtg agcaaaaggc cagcaaaaggccaggaaccg 8400 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacgagcatcacaa 8460 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagataccaggcgtt 8520 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgcttaccggatacct 8580 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgctgtaggtatct 8640 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaaccccccgttcagcc 8700 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaagacacgactt 8760 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatgtaggcggtgc 8820 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacagtatttggtat 8880 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctcttgatccggcaa 8940 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagattacgcgcagaaa 9000 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctcagtggaacga 9060 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttcacctagatcct 9120 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaacttggtctga 9180 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctatttcgttcatc 9240 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggcttaccatctgg 9300 ccccagtgct gcaatgatac cgcgagaacc acgctcaccc gcaccagatttatcagcaat 9360 aaaccagcca gccggaagtg cgctgcggag aagtggtcct gcaactttatccgcctccat 9420 ccagtctatt agttgttgcc gggaagctag agtaagtagt tcgccagtcagcagtttgcg 9480 taacgtcgtt gccatagcaa caggcatcgt ggtgtcacgc tcgtcgtttggtatggcttc 9540 attcagctcc ggctcccaac gatcaaggcg agttacatga tcccccatgttgtgcaaaaa 9600 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccgcagtgttatc 9660 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccgtaagatgctt 9720 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgcggcgaccgag 9780 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaactttaaaagt 9840 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttaccgctgttgag 9900 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatcttttactttcac 9960 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagggaataagggc 10020 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaagcatttatca 10080 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaataaacaaatagg 10140 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaaccattattaccat 10200 gacattaacc tataaaaata ggcgtatcac gaagcccttt cgtctagcgcgtttcggtga 10260 tgacggtgaa aacctctgac acttgcagct cccgcagacg gtcacagcttgtctgtaagc 10320 ggatgccggg agcaggcaag cccgtcaggg cgcgtcagtg ggtgttggcgggtgtcgggg 10380 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtacaccagatgcggtgtga 10440 aataccgcac agatgcgtaa ggagaaaata ccgcatcagc ctccattcgccattcagact 10500 ccgcaactgt tgggaagggc ggtcagtacg cgcttcttcg ctattacgccaactggcgaa 10560 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttcccaatcacgacg 10620 ttgtaaaacg acagccaatg aattgaagct tattaattct agactgaagcttttaatacg 10680 actcactata 10690 4 23 DNA Primer oligonucleotide 4acatgatctg cagagaggcc agt 23 5 26 DNA Primer oligonucleotide 5gacasgctgt gatawatgtc tccccc 26 6 21 DNA Primer oligonucleotide 6tggctctcct caagcgtatt c 21 7 23 DNA Primer oligonucleotide 7 actctctgcagtcaagcggc tca 23 8 21 DNA Primer oligonucleotide 8 cagtggatgaaccggctgat a 21 9 23 DNA Primer oligonucleotide 9 ggggcgacgg catcatgcaaacc 23 10 23 DNA Primer oligonucleotide 10 caggacctgc agtctgtcaa agg 2311 17 DNA Primer oligonucleotide 11 cgggagagcc atagtgg 17 12 19 DNAPrimer oligonucleotide 12 agtaccacaa ggcctttcg 19 13 21 DNA Probe 13ctgcggaacc ggtgagtaca c 21

What is claimed is:
 1. A nucleic acid molecule comprising a regionselected from the group consisting of: a) an altered HCV NS3 encodingregion coding for one or more NS3 mutations, wherein at least one ofsaid NS3 mutations, identified by reference to the amino acid sequencenumbering of SEQ. ID. NO. 1, is selected from the group consisting of:amino acid 1095 being Ala, amino acid 1202 being Gly, and amino acid1347 being Thr; b) an altered HCV NS5A encoding region coding for one ormore NS5A mutations, wherein at least one of said NS5A mutations,identified by reference to the amino acid sequence numbering of SEQ. ID.NO. 1, is selected from the group consisting of: amino acid 2041 beingThr, a Lys insertion between residue 2039 and
 2040. amino acid 2173being Phe, amino acid 2197 being Phe, amino acid 2198 being Ser, aminoacid 2199 being Thr, and amino acid 2204 being Arg; and c) an alteredencephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)region containing one or more EMCV IRES mutations, wherein at least oneof said EMCV IRES mutations, identified by reference to the nucleotidenumber of SEQ. ID. NO. 3, is an insertion at nucleotide 1736 of adenine.2. The nucleic acid molecule of claim 1, wherein said nucleic acidmolecule comprises said NS5A encoding region.
 3. The nucleic acidmolecule of claim 2, wherein at least two of said NS5A adaptivemutations are present.
 4. The nucleic acid molecule of claim 2, furthercomprising a region encoding for a HCV NS3 region, wherein said NS3region may be the same or different than said altered NS3 region.
 5. Thenucleic acid molecule of claim 4, wherein said nucleic acid molecule isan HCV replicon comprising a HCV 5′ UTR-PC region, said NS3 encodingregion, an HCV NS4A encoding region, an HCV NS4B encoding region, saidNS5A encoding region, an HCV NS5B encoding region, and a HCV 3′ UTR. 6.The nucleic acid molecule of claim 5, wherein said HCV replicon furthercomprises a sequence encoding for a reporter protein.
 7. The nucleicacid molecule of claim 5, wherein said HCV replicon further comprises asequence encoding for a selection protein.
 8. The nucleic acid moleculeof claim 5, wherein said HCV replicon further comprises a HCV coreencoding region, a HCV E1 encoding region, a HCV E2 encoding region, aHCV p7 encoding region, and a HCV NS2 encoding region.
 9. A nucleic acidmolecule comprising a region selected from the group consisting of: a)an altered HCV NS3 encoding region containing one or more NS3 mutations,wherein at least one of said NS3 mutations, identified by reference tothe nucleotide numbering of SEQ. ID. NO. 2, is selected from the groupconsisting of: nucleotide 3625 being cytosine, nucleotide 3946 beingguanine, nucleotide 4380 being adenine, b) an altered HCV NS5A encodingregion containing one or more NS5A mutations, wherein at least one ofsaid NS5A mutations, identified by reference to the nucleotide numberingof SEQ. ID. NO. 2, is selected from the group consisting of: aninsertion of 3 adenine residues between nucleotide 6458 and 6459,nucleotide 6463 being cytosine, nucleotide 6859 being thymine or uracil,nucleotide 6931 being thymine or uracil, nucleotide 6934 being cytosine,nucleotide 6936 being adenine, and nucleotide 6953 being adenine orguanine; and c) an altered encephalomyocarditis virus (EMCV) internalribosome entry site (IRES) region containing one or more EMCV IRESmutations, wherein at least one of said EMCV IRES mutations, identifiedby reference to the nucleotide number of SEQ. ID. NO. 3, is an insertionat nucleotide 1736 of adenine.
 10. The nucleic acid molecule of claim 9,wherein said molecule comprises said altered NS5A encoding region, andthe nucleotide sequence of said altered NS5A region is provided for bybases 6258-7598 of SEQ. ID. NO. 2, or the RNA version thereof, modifiedwith one or more of said NS5A modifications selected from the groupconsisting of: an insertion of 3 adenine residues between nucleotide6458 and 6459, nucleotide 6463 being cytosine, nucleotide 6859 beingthymine or uracil, nucleotide 6931 being thymine or uracil, nucleotide6934 being cytosine, nucleotide 6936 being adenine, and nucleotide 6953being adenine or guanine.
 11. The nucleic acid molecule of claim 10,wherein said molecule is an HCV replicon comprising a HCV 5′ UTR-PCregion, a modified HCV NS3-NS5B region, and a HCV 3′ UTR, wherein saidmodified NS3-NS5B region comprises said altered NS5A region.
 12. Thenucleic acid molecule of claim 11, wherein said 5′ UTR-PC region is theRNA version of bases 1-377 of SEQ. ID. NO. 2 and said 3′ UTR is the RNAversion of bases 9374-9605 of SEQ. ID. NO.
 2. 13. The nucleic acidmolecule of claim 10, wherein said molecule is an HCV repliconcomprising a HCV 5′ UTR-PC region, a modified HCV NS3-NS5B region, and aHCV 3′ UTR, wherein said 5′ UTR-PC region is the RNA version of bases1-377 of SEQ. ID. NO. 2; said 3′ UTR is the RNA version of bases9374-9605 of SEQ. ID. NO. 2; and, said modified NS3-NS5B region consistsof the RNA version of bases 3420-9371 of SEQ. ID. NO. 2 modified withone or more modifications selected from the group consisting of:nucleotide 4380 being adenine, nucleotide 3625 being cytosine,nucleotide 3946 being guanine, an insertion of 3 adenine residuesbetween nucleotide 6458 and nucleotide 6459, nucleotide 6463 beingcytosine, nucleotide 6859 being uracil, nucleotide 6931 being uracil,nucleotide 6934 being cytosine, nucleotide 6936 being adenine, andnucleotide 6953 being adenine or guanine.
 14. The nucleic acid moleculeof claim 13, wherein said replicon is a genomic replicon that furthercomprises the RNA version of nucleotides 378-3419 of SEQ. ID. NO.
 2. 15.A nucleic acid molecule comprising the nucleic acid base sequence ofbases 1-7989 of SEQ. ID. NO. 3, or the RNA version thereof, consistingof one or more different modifications selected from the groupconsisting of: a) nucleotides 5335-5337 modified to code for arginine;b) nucleotides 5242-5244 modified to code for phenylalanine; c)nucleotides 5314-5316 modified to code for phenylalanine; d) nucleotides5317-5319 modified to code for serine; e) nucleotides coding for lysineinserted after nucleotide 4843; f) nucleotides 2329-2331 modified tocode for glycine, nucleotides 2764-2766 modified to code for threonine,nucleotides 5242-5244 modified to code for phenylalanine, and an extraadenosine inserted after nucleotide 1736; g) nucleotides 4846-4848modified to code for threonine, and nucleotides 5242-5244 modified tomodified to code for phenylalanine; h) nucleotides 4846-4848 modified tocode for threonine, and nucleotides 5314-5316 modified to code forphenylalanine; i) nucleotides 4846-4848 modified to code for threonine,and nucleotides 5317-5319 modified to code for serine; j) nucleotides2329-2331 modified to code for glycine, and nucleotides coding forlysine inserted after nucleotides 4843; k) nucleotides 5314-5316modified to code for phenylalanine and nucleotides 5320-5322 modified tocode for threonine; l) nucleotides 4846-4848 modified to code forthreonine, nucleotides 5314-5316 modified to code for phenylalanine, andnucleotides 5320-5322 modified to code for threonine; m) nucleotides4846-4848 modified to code for threonine, nucleotides 5314-5316 modifiedto code for phenylalanine, and an extra adenosine inserted afternucleotide 1736; and n) nucleotides 5314-5316 modified to code forphenylalanine, nucleotides 5320-5322 modified to code for threonine, andan extra adenosine inserted after nucleotide 1736; and o) nucleotides5320-5322 modified to code for threonine.
 16. The nucleic acid of claim15, wherein said one or more different modifications is selected fromthe group consisting of: a) C5337A; b) C5243T or U; c) C5315T or U; d) Tor U5318C; e) AAA inserted after 4843; f) A2330G, G2764A, C5243T or U,and adenosine inserted 1736; g) A4847C and C5243T or U; h) A4847C andC5315T or U; i) A4847C and T or U5318C; j) A2330G and AAA inserted after4843; k) C5315T or U and G5320A; l) A4847C, C5315T or U, and G5320A; m)A4847C, C5315T or U, and adenosinc inserted 1736; n) C5315T or U, G5320Aand adenosine inserted 1736; and o) G5320A.
 17. The nucleic acid ofclaim 16, wherein said nucleic acid is RNA and comprises said nucleicacid base sequence.
 18. The nucleic acid of claim 17, wherein saidnucleic acid is RNA and consists of said nucleic acid base sequence. 19.An expression vector comprising a nucleotide sequence coding for thenucleic acid molecule of any one of claims 1-18, wherein said nucleotidesequence is transcriptionally coupled to an exogenous promoter.
 20. Arecombinant cell human hepatoma cell, wherein said cell comprises thenucleic acid of any one of claims 5-8 and 11-18.
 21. The recombinantcell of claim 20, wherein said hepatoma cell is an Huh-7 cell.
 22. Therecombinant cell of claim 20, wherein said cell is derived from a Huh-7cell.
 23. A recombinant cell made by a process comprising the step ofintroducing into a human hepatoma cell the nucleic acid of any one ofclaims 5-8 and 11-18.
 24. A method of making an HCV replicon enhancedcell comprising the steps of: a) introducing and maintaining a HCVreplicon in a cell; and b) curing said cell of said HCV replicon toproduce said replicon enhanced cell.
 25. The method of claim 24, whereinsaid cell is a human hepatoma cell.
 26. The method of claim 24, whereinsaid cell is a Huh-7 cell or is derived from a Huh-7 cell.
 27. Themethod of claim 26, further comprising the step of confirming theability of said replicon enhanced cell to maintain an HCV replicon. 28.A method of making an HCV replicon enhanced cell containing a functionalHCV replicon comprising the steps of: a) introducing and maintaining afirst HCV replicon in a cell; b) curing said cell of said first repliconto produce a cured cell; and c) introducing and maintaining a second HCVreplicon into said cured cell, wherein said second HCV replicon may bethe same or different than said first HCV replicon.
 29. The method ofclaim 28, wherein said cell is a human hepatoma cell.
 30. The method ofclaim 29, wherein said human hepatoma cell is a Huh-7 cell.
 31. Themethod of claim 30, wherein said human hepatoma cell is derived from aHuh-7 cell.
 32. An HCV replicon enhanced cell made by the method of anyone of claims 24-27.
 33. An HCV replicon enhanced cell containing a HCVreplicon made by the method of any one of claims 28-31.
 34. A method ofmeasuring the ability of a compound to affect HCV activity comprisingthe steps of: a) providing said compound to the HCV replicon enhancedcell of claim 33; and b) measuring the ability of said compound toeffect one or more replicon activities as a measure of the effect on HCVactivity.
 35. The method of claim 34, wherein said compound is aribozyme.
 36. The method of claim 34, wherein said compound in anantisense nucleic acid.
 37. The method of claim 34, wherein compound isan organic compound.
 38. The method of claim 34, wherein said step (b)measures HCV protein production.
 39. The method of claim 33, whereinsaid step (b) measures production of RNA transcripts.